Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects.

Neuronal precursors, generated throughout life in the subventricular zone, migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. We found that the PI3K-Akt-mTorc1 pathway is selectively inactivated in migrating neuroblasts in the subventricular zone and rostral migratory stream, and activated when these cells reach the olfactory bulb. Postnatal deletion of Pten caused aberrant activation of the PI3K-Akt-mTorc1 pathway and an enlarged subventricular zone and rostral migratory stream. This expansion was caused by premature termination of migration and differentiation of neuroblasts and was rescued by inhibition of mTorc1. This phenotype is reminiscent of lamination defects caused by Pten deletion in developing brain that were previously described as defective migration. However, live imaging in acute slices showed that Pten deletion did not cause a uniform defect in the mechanics of directional neuroblast migration. Instead, a subpopulation of Pten-null neuroblasts showed minimal movement and altered morphology associated with differentiation, whereas the remainder showed unimpeded directional migration towards the olfactory bulb. Therefore, migration defects of Pten-null neurons might be secondary to ectopic differentiation.

A perivascular niche for brain tumor stem cells.

Cancers are believed to arise from cancer stem cells (CSCs), but it is not known if these cells remain dependent upon the niche microenvironments that regulate normal stem cells. We show that endothelial cells interact closely with self-renewing brain tumor cells and secrete factors that maintain these cells in a stem cell-like state. Increasing the number of endothelial cells or blood vessels in orthotopic brain tumor xenografts expanded the fraction of self-renewing cells and accelerated the initiation and growth of tumors. Conversely, depletion of blood vessels from xenografts ablated self-renewing cells from tumors and arrested tumor growth. We propose that brain CSCs are maintained within vascular niches that are important targets for therapeutic approaches.

Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1(C-L)), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1(+/C-L) mice containing the Rosa26-YFP reporter allele showed that Prom1(+) cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1(+/C-L) mice containing a Cre-dependent mutant allele of beta-catenin (Ctnnb1(lox(ex3))) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1(+) cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1(+) cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1(+) tumour cells.

Forward suppression in the auditory cortex is caused by the Ca(v)3.1 calcium channel-mediated switch from bursting to tonic firing at thalamocortical projections.

Brief sounds produce a period of suppressed responsiveness in the auditory cortex (ACx). This forward suppression can last for hundreds of milliseconds and might contribute to mechanisms of temporal separation of sounds and stimulus-specific adaptation. However, the mechanisms of forward suppression remain unknown. We used in vivo recordings of sound-evoked responses in the mouse ACx and whole-cell recordings, two-photon calcium imaging in presynaptic terminals, and two-photon glutamate uncaging in dendritic spines performed in brain slices to show that synaptic depression at thalamocortical (TC) projections contributes to forward suppression in the ACx. Paired-pulse synaptic depression at TC projections lasts for hundreds of milliseconds and is attributable to a switch between firing modes in thalamic neurons. Thalamic neurons respond to a brief depolarizing pulse with a burst of action potentials; however, within hundreds of milliseconds, the same pulse repeated again produces only a single action potential. This switch between firing modes depends on Ca(v)3.1 T-type calcium channels enriched in thalamic relay neurons. Pharmacologic inhibition or knockdown of Ca(v)3.1 T-type calcium channels in the auditory thalamus substantially reduces synaptic depression at TC projections and forward suppression in the ACx. These data suggest that Ca(v)3.1-dependent synaptic depression at TC projections contributes to mechanisms of forward suppression in the ACx.

Thalamocortical long-term potentiation becomes gated after the early critical period in the auditory cortex.

Cortical maps in sensory cortices are plastic, changing in response to sensory experience. The cellular site of such plasticity is currently debated. Thalamocortical (TC) projections deliver sensory information to sensory cortices. TC synapses are currently dismissed as a locus of cortical map plasticity because TC synaptic plasticity is thought to be limited to neonates, whereas cortical map plasticity can be induced in both neonates and adults. However, in the auditory cortex (ACx) of adults, cortical map plasticity can be induced if animals attend to a sound or receive sounds paired with activation of cholinergic inputs from the nucleus basalis. We now show that, in the ACx, long-term potentiation (LTP), a major form of synaptic plasticity, is expressed at TC synapses in both young and mature mice but becomes gated with age. Using single-cell electrophysiology, two-photon glutamate uncaging, and optogenetics in TC slices containing the auditory thalamus and ACx, we show that TC LTP is expressed postsynaptically and depends on group I metabotropic glutamate receptors. TC LTP in mature ACx can be unmasked by cortical disinhibition combined with activation of cholinergic inputs from the nucleus basalis. Cholinergic inputs passing through the thalamic radiation activate M1 muscarinic receptors on TC projections and sustain glutamate release at TC synapses via negative regulation of presynaptic adenosine signaling through A1 adenosine receptors. These data indicate that TC LTP in the ACx persists throughout life and therefore can potentially contribute to experience-dependent cortical map plasticity in the ACx in both young and adult animals.

Retinoblastoma (Rb) regulates laminar dendritic arbor reorganization in retinal horizontal neurons.

Neuronal differentiation with respect to the acquisition of synaptic competence needs to be regulated precisely during neurogenesis to ensure proper formation of circuits at the right place and time in development. This regulation is particularly important for synaptic triads among photoreceptors, horizontal cells (HCs), and bipolar cells in the retina, because HCs are among the first cell types produced during development, and bipolar cells are among the last. HCs undergo a dramatic transition from vertically oriented neurites that form columnar arbors to overlapping laminar dendritic arbors with differentiation. However, how this process is regulated and coordinated with differentiation of photoreceptors and bipolar cells remains unknown. Previous studies have suggested that the retinoblastoma (Rb) tumor suppressor gene may play a role in horizontal cell differentiation and synaptogenesis. By combining genetic mosaic analysis of individual synaptic triads with neuroanatomic analyses and multiphoton live imaging of developing HCs, we found that Rb plays a cell-autonomous role in the reorganization of horizontal cell neurites as they differentiate. Aberrant vertical processes in Rb-deficient HCs form ectopic synapses with rods in the outer nuclear layer but lack bipolar dendrites. Although previous reports indicate that photoreceptor abnormalities can trigger formation of ectopic synapses, our studies now demonstrate that defects in a postsynaptic partner contribute to the formation of ectopic photoreceptor synapses in the mammalian retina.

Sound-evoked adenosine release in cooperation with neuromodulatory circuits permits auditory cortical plasticity and perceptual learning.

Meaningful auditory memories are formed in adults when acoustic information is delivered to the auditory cortex during heightened states of attention, vigilance, or alertness, as mediated by neuromodulatory circuits. Here, we identify that, in awake mice, acoustic stimulation triggers auditory thalamocortical projections to release adenosine, which prevents cortical plasticity (i.e., selective expansion of neural representation of behaviorally relevant acoustic stimuli) and perceptual learning (i.e., experience-dependent improvement in frequency discrimination ability). This sound-evoked adenosine release (SEAR) becomes reduced within seconds when acoustic stimuli are tightly paired with the activation of neuromodulatory (cholinergic or dopaminergic) circuits or periods of attentive wakefulness. If thalamic adenosine production is enhanced, then SEAR elevates further, the neuromodulatory circuits are unable to sufficiently reduce SEAR, and associative cortical plasticity and perceptual learning are blocked. This suggests that transient low-adenosine periods triggered by neuromodulatory circuits permit associative cortical plasticity and auditory perceptual learning in adults to occur.

Synaptic plasticity in human thalamocortical assembloids.

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Synaptic plasticity in human thalamocortical assembloids.

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Presynaptic gating of postsynaptically expressed plasticity at mature thalamocortical synapses.

Thalamocortical (TC) projections provide the major pathway for ascending sensory information to the mammalian neocortex. Arrays of these projections form synaptic inputs on thalamorecipient neurons, thus contributing to the formation of receptive fields (RFs) in sensory cortices. Experience-dependent plasticity of RFs persists throughout an organism's life span but in adults requires activation of cholinergic inputs to the cortex. In contrast, synaptic plasticity at TC projections is limited to the early postnatal period. This disconnect led to the widespread belief that TC synapses are the principal site of RF plasticity only in neonatal sensory cortices, but that they lose this plasticity upon maturation. Here, we tested an alternative hypothesis that mature TC projections do not lose synaptic plasticity but rather acquire gating mechanisms that prevent the induction of synaptic plasticity. Using whole-cell recordings and direct measures of postsynaptic and presynaptic activity (two-photon glutamate uncaging and two-photon imaging of the FM 1-43 assay, respectively) at individual synapses in acute mouse brain slices that contain the auditory thalamus and cortex, we determined that long-term depression (LTD) persists at mature TC synapses but is gated presynaptically. Cholinergic activation releases presynaptic gating through M(1) muscarinic receptors that downregulate adenosine inhibition of neurotransmitter release acting through A(1) adenosine receptors. Once presynaptic gating is released, mature TC synapses can express LTD postsynaptically through group I metabotropic glutamate receptors. These results indicate that synaptic plasticity at TC synapses is preserved throughout the life span and, therefore, may be a cellular substrate of RF plasticity in both neonate and mature animals.

Phosphatase and tensin homologue (PTEN) regulates synaptic plasticity independently of its effect on neuronal morphology and migration.

The tumour suppressor PTEN is the central negative regulator of the phosphatidylinositol 3-kinase (PI3K) signalling pathway, which mediates diverse processes in various tissues. In the nervous system, the PI3K pathway modulates proliferation, migration, cellular size, synaptic transmission and plasticity. In humans, neurological abnormalities such as autism, seizures and ataxia are associated with inherited PTEN mutations. In rodents, Pten loss during early development is associated with extensive deficits in neuronal migration and substantial hypertrophy of neurons and synaptic densities; however, whether its effect on synaptic transmission and plasticity is direct or mediated by structural abnormalities remains unknown. Here we analysed neuronal and synaptic structures and function in Pten-conditional knockout mice in which the gene was deleted from excitatory neurons postnatally. Using two-photon imaging, Golgi staining, immunohistochemistry, electron microscopy, and electrophysiological tools, we determined that Pten loss does not affect hippocampus development, neuronal or synaptic structures, or basal excitatory synaptic transmission. However, it does cause deficits in both major forms of synaptic plasticity, long-term potentiation and long-term depression, of excitatory synaptic transmission. These deficits coincided with impaired spatial memory, as measured in water maze tasks. Deletion of Pdk1, which encodes a positive downstream regulator of the PI3K pathway, rescued Pten-mediated deficits in synaptic plasticity but not in spatial memory. These results suggest that PTEN independently modulates functional and structural properties of hippocampal neurons and is directly involved in mechanisms of synaptic plasticity.

Dysregulation of presynaptic calcium and synaptic plasticity in a mouse model of 22q11 deletion syndrome.

The 22q11 deletion syndrome (22q11DS) is characterized by cognitive decline and increased risk of psychiatric disorders, mainly schizophrenia. The molecular mechanisms of neuronal dysfunction in cognitive symptoms of 22q11DS are poorly understood. Here, we report that a mouse model of 22q11DS, the Df(16)1/+ mouse, exhibits substantially enhanced short- and long-term synaptic plasticity at hippocampal CA3-CA1 synapses, which coincides with deficits in hippocampus-dependent spatial memory. These changes are evident in mature but not young animals. Electrophysiological, two-photon imaging and glutamate uncaging, and electron microscopic assays in acute brain slices showed that enhanced neurotransmitter release but not altered postsynaptic function or structure caused these changes. Enhanced neurotransmitter release in Df(16)1/+ mice coincided with altered calcium kinetics in CA3 presynaptic terminals and upregulated sarco(endo)plasmic reticulum calcium-ATPase type 2 (SERCA2). SERCA inhibitors rescued synaptic phenotypes of Df(16)1/+ mice. Thus, presynaptic SERCA2 upregulation may be a pathogenic event contributing to the cognitive symptoms of 22q11DS.

Connectivity patterns revealed by mapping of active inputs on dendrites of thalamorecipient neurons in the auditory cortex.

Despite being substantially outnumbered by intracortical inputs on thalamorecipient neurons, thalamocortical projections efficiently deliver acoustic information to the auditory cortex. We hypothesized that thalamic projections may achieve effectiveness by forming synapses at optimal locations on dendritic trees of cortical neurons. Using two-photon calcium imaging in dendritic spines, we constructed maps of active thalamic and intracortical inputs on dendritic trees of thalamorecipient cortical neurons in mouse thalamocortical slices. These maps revealed that thalamic projections synapse preferentially on stubby dendritic spines within 100 microm of the soma, whereas the locations and morphology of spines that receive intracortical projections have a less-defined pattern. Using two-photon photolysis of caged glutamate, we found that activation of stubby dendritic spines located perisomatically generated larger postsynaptic potentials in the soma of thalamorecipient neurons than did activation of remote dendritic spines or spines of other morphological types. These results suggest a novel mechanism of reliability of thalamic projections: the positioning of crucial afferent inputs at optimal synaptic locations.

Orphan glutamate receptor delta1 subunit required for high-frequency hearing.

The function of the orphan glutamate receptor delta subunits (GluRdelta1 and GluRdelta2) remains unclear. GluRdelta2 is expressed exclusively in the Purkinje cells of the cerebellum, and GluRdelta1 is prominently expressed in inner ear hair cells and neurons of the hippocampus. We found that mice lacking the GluRdelta1 protein displayed significant cochlear threshold shifts for frequencies of >16 kHz. These deficits correlated with a substantial loss of type IV spiral ligament fibrocytes and a significant reduction of endolymphatic potential in high-frequency cochlear regions. Vulnerability to acoustic injury was significantly enhanced; however, the efferent innervation of hair cells and the classic efferent inhibition of outer hair cells were unaffected. Hippocampal and vestibular morphology and function were normal. Our findings show that the orphan GluRdelta1 plays an essential role in high-frequency hearing and ionic homeostasis in the basal cochlea, and the locus encoding GluRdelta1 represents a candidate gene for congenital or acquired high-frequency hearing loss in humans.

Slow presynaptic and fast postsynaptic components of compound long-term potentiation.

Long-term potentiation (LTP) mediates learning and memory in the mammalian hippocampus. Whether a presynaptic or postsynaptic neuron principally enhances synaptic transmission during LTP remains controversial. Acute hippocampal slices were made from transgenic mouse strains that express synaptopHluorin in neurons. SynaptopHluorin is an indicator of synaptic vesicle recycling; thus, we monitored functional changes in presynaptic boutons of CA3 pyramidal cells by measuring changes in synaptopHluorin fluorescence. Simultaneously, we recorded field excitatory postsynaptic potentials to monitor changes in the strength of excitatory synapses between CA3 and CA1 pyramidal neurons. We found that LTP consists of two components, a slow presynaptic component and a fast postsynaptic component. The presynaptic mechanisms contribute mostly to the late phase of compound LTP, whereas the postsynaptic mechanisms are crucial during the early phase of LTP. We also found that protein kinase A (PKA) and L-type voltage-gated calcium channels are crucial for the expression of the presynaptic component of compound LTP, and NMDA channels are essential for that of the postsynaptic component of LTP. These data are the first direct evidence that presynaptic and postsynaptic components of LTP are temporally and mechanistically distinct.

Rab-mediated endocytosis: linking neurodegeneration, neuroprotection, and synaptic plasticity?

Rab proteins are small GTPases involved in endocytosis and recycling of cell surface molecules. Recently they have been implicated in the etiopathogenesis of several neurodegenerative disorders including Alzheimer's and Lewy body disease. In experiments on organotypic hippocampal cultures, upregulation of Rab protein family member Rab5b after group I metabotropic glutamate receptor (mGluR) stimulation was associated with reduced neuronal vulnerability to excitotoxic injury. This mGluR-mediated neuroprotection was abolished by antisense-induced deficiency of Rab5b. Electrophysiological measurements of excitatory synaptic transmission in the Schaffer collateral-CA1 pathway revealed that mGluR activation that induces neuroprotection also induced long-term depression (LTD) of synaptic transmission. Similar to the neuroprotection, Rab5b deficiency abolished dihydroxyphenylglycine-induced LTD. Together, these findings support the idea that Rab proteins, and the Rab5b protein in particular, may provide a link between neurodegenerative disease, neuroprotection, and synaptic plasticity, as well as possibly being a useful target for pharmacological interventions.

Schizophrenia-Related Microdeletion Impairs Emotional Memory through MicroRNA-Dependent Disruption of Thalamic Inputs to the Amygdala.

Individuals with 22q11.2 deletion syndrome (22q11DS) are at high risk of developing psychiatric diseases such as schizophrenia. Individuals with 22q11DS and schizophrenia are impaired in emotional memory, anticipating, recalling, and assigning a correct context to emotions. The neuronal circuits responsible for these emotional memory deficits are unknown. Here, we show that 22q11DS mouse models have disrupted synaptic transmission at thalamic inputs to the lateral amygdala (thalamo-LA projections). This synaptic deficit is caused by haploinsufficiency of the 22q11DS gene Dgcr8, which is involved in microRNA processing, and is mediated by the increased dopamine receptor Drd2 levels in the thalamus and by reduced probability of glutamate release from thalamic inputs. This deficit in thalamo-LA synaptic transmission is sufficient to cause fear memory deficits. Our results suggest that dysregulation of the Dgcr8-Drd2 mechanism at thalamic inputs to the amygdala underlies emotional memory deficits in 22q11DS.

Thalamic miR-338-3p mediates auditory thalamocortical disruption and its late onset in models of 22q11.2 microdeletion.

Although 22q11.2 deletion syndrome (22q11DS) is associated with early-life behavioral abnormalities, affected individuals are also at high risk for the development of schizophrenia symptoms, including psychosis, later in life. Auditory thalamocortical (TC) projections recently emerged as a neural circuit that is specifically disrupted in mouse models of 22q11DS (hereafter referred to as 22q11DS mice), in which haploinsufficiency of the microRNA (miRNA)-processing-factor-encoding gene Dgcr8 results in the elevation of the dopamine receptor Drd2 in the auditory thalamus, an abnormal sensitivity of thalamocortical projections to antipsychotics, and an abnormal acoustic-startle response. Here we show that these auditory TC phenotypes have a delayed onset in 22q11DS mice and are associated with an age-dependent reduction of miR-338-3p, a miRNA that targets Drd2 and is enriched in the thalamus of both humans and mice. Replenishing depleted miR-338-3p in mature 22q11DS mice rescued the TC abnormalities, and deletion of Mir338 (which encodes miR-338-3p) or reduction of miR-338-3p expression mimicked the TC and behavioral deficits and eliminated the age dependence of these deficits. Therefore, miR-338-3p depletion is necessary and sufficient to disrupt auditory TC signaling in 22q11DS mice, and it may mediate the pathogenic mechanism of 22q11DS-related psychosis and control its late onset.

Group I metabotropic glutamate receptors reduce excitotoxic injury and may facilitate neurogenesis.

Group I metabotropic glutamate receptor (mGluR) agonist DHPG reduced nerve cell death caused by their exposure to NMDA ("neuroprotective effect") and attenuated NMDA receptor-mediated currents [Blaabjerg, M., Baskys, A., Zimmer, J., Vawter, M. P., 2003b. Changes in hippocampal gene expression after neuroprotective activation of group I metabotropic glutamate receptors. Brain Research, Molecular Brain Research 117, 196-205.]. In the present study, we used organotypic hippocampal culture preparation to examine specific phospholipase C (PLC) inhibitor U73122 effects on DHPG-induced neuroprotection, changes in excitatory synaptic transmission associated with the neuroprotective DHPG treatment and a role of group I mGluR ligands in neurogenesis. Results show that short (10 min) DHPG treatment did not result in neuroprotection but significantly depressed field synaptic potentials (fEPSP) in the Schaffer collateral-CA1 pathway. The fEPSP depression was not affected by the PLC inhibitor U73122. In contrast, prolonged (2-h) treatment of cultures with DHPG induced a significant protective effect that was blocked by a PLC inhibitor U73122 but not by its inactive analog U73343. Voltage-clamp measurements of spontaneous miniature excitatory post-synaptic currents (EPSCs) recorded in CA1 neurons from cultures treated with DHPG (10 microM, 2 h) showed a significant reduction of the EPSC amplitude in DHPG-treated but not control (untreated) cultures. This reduction was completely abolished by U73122, suggesting a PLC involvement. Since activation of PLC is thought to be associated with cell proliferation, we investigated whether group I mGluR agonist DHPG or subtype antagonists LY367385 and MPEP have an effect on dentate granule cells expressing immature neuronal marker TOAD-64. DHPG (100 microM, 72 h) slightly but not significantly increased the number of TOAD-64 positive cells. The mGluR1 antagonists LY367385 (10 microM, 72 h) markedly decreased the number of TOAD-64 positive cells and mGluR5 antagonist MPEP (1 microM, 72 h) had no effect. These data suggest that (1) prolonged activation of group I mGluRs reduces nerve cell susceptibility to excitotoxic injury in a PLC-dependent manner; (2) this reduction is associated with a PLC-dependent depression of excitatory synaptic transmission; and (3) mGluR1 activation may facilitate neurogenesis.

Activation of neuroprotective pathways by metabotropic group I glutamate receptors: a potential target for drug discovery?

Stroke neuroprotection trials suggest that pharmacological manipulations of a single neuroprotective mechanism are generally ineffective and that new approaches, possibly involving simultaneous manipulations of multiple mechanisms, need to be sought. To identify optimal components for such a multipronged approach, we studied NMDA receptor activation-induced cell death in organotypic hippocampal culture preparations as a model of excitotoxicity. Metabotropic group I glutamate receptor (mGluR) activation by their selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), resulted in concentration-dependent reduction of nerve cell susceptibility to NMDA-mediated injury (neuroprotective effect). The neuroprotection was mediated primarily by mGluR1, required phospholipase C activation, was inhibited by cholesterol-containing methyl-beta-cyclodextrin treatment, and occluded by antipsychotic quetiapine. It was associated with suppression of NMDA currents and prolongation of GABA(A) receptor-mediated currents in DHPG-treated cultures. cDNA microarray analysis of 1128 brain-relevant genes revealed that mGluR-mediated neuroprotection was associated with simultaneous activation of endocytosis, and inactivation of inflammation, cell adhesion, cell death, and transcription-related genes. Antisense inhibition of Rab5b, a gene coding for a small GTPase associated with endocytosis, significantly reduced the mGluR-mediated neuroprotection. These findings expand our understanding of the role that mGluRs play in regulation of nerve cell susceptibility to injury and should facilitate the design of novel therapeutic strategies for stroke and other neurodegenerative diseases.