Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development.

The development of glutamatergic synapses involves changes in the number and type of receptors present at the postsynaptic density. To elucidate molecular mechanisms underlying these changes, we combine in utero electroporation of constructs that alter the molecular composition of developing synapses with dual whole-cell electrophysiology to examine synaptic transmission during two distinct developmental stages. We find that SAP102 mediates synaptic trafficking of AMPA and NMDA receptors during synaptogenesis. Surprisingly, after synaptogenesis, PSD-95 assumes the functions of SAP102 and is necessary for two aspects of synapse maturation: the developmental increase in AMPA receptor transmission and replacement of NR2B-NMDARs with NR2A-NMDARs. In PSD-95/PSD-93 double-KO mice, the maturational replacement of NR2B- with NR2A-NMDARs fails to occur, and PSD-95 expression fully rescues this deficit. This study demonstrates that SAP102 and PSD-95 regulate the synaptic trafficking of distinct glutamate receptor subtypes at different developmental stages, thereby playing necessary roles in excitatory synapse development.

Characterization of mice with targeted deletion of glycine receptor alpha 2.

Glycine receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system. During development, glycine receptor alpha 2 (GlyRalpha2) is expressed in the retina, in the spinal cord, and throughout the brain. Within the cortex, GlyRalpha2 is expressed in immature cells and these receptors have been shown to be active and excitatory. In the developing retina, inhibition of glycine receptor activity prevents proper rod photoreceptor development. These data suggest that GlyRalpha2, the developmentally expressed glycine receptor, may play an important role in neuronal development. We have generated mice with a targeted deletion of glycine receptor alpha 2 (Glra2). Although these mice lack expression of GlyRalpha2, no gross morphological or molecular alterations were observed in the nervous system. In addition, the cerebral cortex does not appear to require glycine receptor activity for proper development, as Glra2 knockout mice did not show any electrophysiological responses to glycine.

GABA may act as a self-limiting trophic factor at developing synapses.

Early in development, synapses with glycine or gamma-aminobutyric acid (GABA)-gated chloride channels exhibit the ability to depolarize postsynaptic cells. As the synapses mature and the gradient of chloride ions across the cell membrane is altered, these neurotransmitters signal an inhibitory response, hyperpolarizing the membrane and decreasing neuronal excitability. Kriegstein and Owens discuss how GABA-stimulated up-regulation of the expression of the potassium chloride cotransporter KCC2 may be the mechanism underlying this synaptic switch.

Neurons derived from radial glial cells establish radial units in neocortex.

The neocortex of the adult brain consists of neurons and glia that are generated by precursor cells of the embryonic ventricular zone. In general, glia are generated after neurons during development, but radial glia are an exception to this rule. Radial glia are generated before neurogenesis and guide neuronal migration. Radial glia are mitotically active throughout neurogenesis, and disappear or become astrocytes when neuronal migration is complete. Although the lineage relationships of cortical neurons and glia have been explored, the clonal relationship of radial glia to other cortical cells remains unknown. It has been suggested that radial glia may be neuronal precursors, but this has not been demonstrated in vivo. We have used a retroviral vector encoding enhanced green fluorescent protein to label precursor cells in vivo and have examined clones 1-3 days later using morphological, immunohistochemical and electrophysiological techniques. Here we show that clones consist of mitotic radial glia and postmitotic neurons, and that neurons migrate along clonally related radial glia. Time-lapse images show that proliferative radial glia generate neurons. Our results support the concept that a lineage relationship between neurons and proliferative radial glia may underlie the radial organization of neocortex.

An excitatory GABAergic plexus in developing neocortical layer 1.

Layer 1 of the developing rodent somatosensory cortex contains a dense, transient GABAergic fiber plexus. Axons arising from the zona incerta (ZI) of the ventral thalamus contribute to this plexus, as do axons of intrinsic GABAergic cells of layer 1. The function of this early-appearing fiber plexus is not known, but these fibers are positioned to contact the apical dendrites of most postmigratory neurons. Here we show that electrical stimulation of layer 1 results in a GABA(A)-mediated postsynaptic current (PSC) in pyramidal neurons. Gramicidin perforated patch recording demonstrates that the GABAergic layer 1 synapse is excitatory and can trigger action potentials in cortical neurons. In contrast to electrical stimulation, activation of intrinsic layer 1 neurons with a glutamate agonist fails to produce PSCs in pyramidal cells. In addition, responses can be evoked by stimulation of layer 1 at relatively large distances from the recording site. These findings are consistent with a contribution of the widely projecting incertocortical pathway, the only described GABAergic projection to neonatal cortex. Recording of identified neonatal incertocortical neurons reveals a population of active cells that exhibit high frequencies of spontaneous action potentials and are capable of robustly activating neonatal cortical neurons. Because the fiber plexus is confined to layer 1, this pathway provides a spatially restricted excitatory GABAergic innervation of the distal apical dendrites of pyramidal neurons during the peak period of cortical synaptogenesis.

Extrinsic GABAergic innervation of developing neocortical layer 1 in organotypic slice co-cultures.

Afferents from the zona incerta (ZI) of the ventral thalamus contribute to the dense, transient gamma-aminobutyric acid (GABA)ergic fiber plexus in layer 1 of the developing rodent somatosensory cortex. Incertocortical axons contact the distal apical dendrites of postmigratory cortical pyramidal cells. Although recent work has shown that these GABAergic incertocortical fibers are likely to provide widespread fast synaptic excitation of pyramidal cells in layers 2-6 during peak periods of cortical synaptogenesis, little is known about the mechanisms by which these axons project to the neocortex and are confined to layer 1. Here we characterize organotypic slice co-cultures in which a region of embryonic diencephalon containing the ZI is maintained adjacent to a region of embryonic somatosensory cortex. Diencephalic explants from transgenic mice expressing enhanced green fluorescent protein (EGFP) enabled direct visualization of diencephalocortical connections. Isochronic co-cultures exhibited diencephalocortical fiber ingrowth immunoreactive for both GABA and the presynaptic vesicle-associated protein synaptophysin that was restricted to neocortical layer 1. This pattern of lamina-specific diencephalocortical ingrowth occurred irrespective of placement of the afferent explant, and persisted in the absence of action potential activity and GABA(A) receptor activation. Heterochronic co-cultures containing older cortex demonstrated that the cortical explants remain permissive for lamina-specific ingrowth through the first postnatal week. Organotypic slice cocultures provide a system in which to study the mechanisms underlying the layer 1-specific ingrowth of extrinsic GABAergic inputs to the perinatal neocortex.

Calcium dynamics of neocortical ventricular zone cells.

Cell-cell signaling within the neocortical ventricular zone (VZ) has been shown to influence the proliferation of VZ precursor cells and the subsequent differentiation and fate of postmitotic neurons. Calcium (Ca(2+)), a ubiquitous second messenger implicated in the regulation of many aspects of development, may play a role in these signaling events. Accordingly, we have examined the spatiotemporal patterns of spontaneous intracellular free Ca(2+) ([Ca(2+)](i)) fluctuations of cells within the intact neocortical VZ. Previous observations have demonstrated that similar patterns of spontaneous [Ca(2+)](i) increase occur in both proliferative and postmitotic cortical cells, suggesting that they may be mechanistically similar. Our results suggest that the changes in [Ca(2+)](i) in VZ cells and cortical plate neurons are likely triggered by different mechansims, and imply that similar changes in [Ca(2+)](i) may underlie different signaling events during distinct phases of neocortical development.

Leukoencephalopathy and raised brain lactate from heroin vapor inhalation ("chasing the dragon")

BACKGROUND: Inhalation of heated heroin vapor ("chasing the dragon"), which is gaining popularity among drug users seeking to avoid the risks of parenteral drug administration, can produce progressive spongiform leukoencephalopathy. METHODS: We studied the clinical phenotype and course, MRI, MRS, and brain pathology in the first American patients described with this syndrome. RESULTS: Two of the three heroin users studied inhaled heroin pyrolysate together daily over the course of 2 weeks. They developed ataxia, dysmetria, and dysarthria. Patient 1 progressed to an akinetic mute state with decorticate posture and subsequent spastic quadriparesis. Patient 2 developed a mild spastic quadriparesis and gait freezing. Patient 3 was asymptomatic following less heroin exposure. Brain MRI showed diffuse, symmetrical white matter hyperintensities in the cerebellum, posterior cerebrum, posterior limbs of the internal capsule, splenium of the corpus callosum, medial lemniscus, and lateral brainstem. MRS showed elevated lactate. Brain biopsy (Patient 1) showed white matter spongiform degeneration with relative sparing of U-fibers; electron microscopy revealed intramyelinic vacuolation with splitting of intraperiod lines. Progressive deterioration occurred in Patients 1 and 2 over 4 weeks. Both were treated with antioxidants including oral coenzyme Q, and clinical improvement occurred. Patient 1 recovered nearly completely over 24 months. Patient 2 improved, but developed a delayed-onset cerebellar hand tremor. Both still have white matter abnormalities on MRI and MRS. CONCLUSIONS: Elevated lactate in white matter and the possible response to antioxidants suggests mitochondrial dysfunction in progressive spongiform leukoencephalopathy following inhalation of heated heroin vapor.

Endogenous activation of metabotropic glutamate receptors in neocortical development causes neuronal calcium oscillations.

Oscillations in intracellular free calcium concentration ([Ca(2+)](i)) occur spontaneously in immature neurons of the developing cerebral cortex. Here, we show that developing murine cortical neurons exhibit calcium oscillations in response to direct activation of the mGluR5 subtype of the group I metabotropic glutamate receptor (mGluR). In contrast, other manipulations that elicit [Ca(2+)](i) increases produce simple, nonoscillatory changes. Furthermore, we find that spontaneous oscillatory [Ca(2+)](i) activity is blocked by antagonists of group I mGluRs, suggesting a specific role for mGluR activation in the promotion of oscillatory [Ca(2+)](i) dynamics in immature cortical neurons. The oscillatory pattern of [Ca(2+)](i) increases produced by mGluR activation might play a role in the regulation of gene expression and the control of developmental events.

Changing properties of GABA(A) receptor-mediated signaling during early neocortical development.

Evidence from several brain regions suggests gamma-aminobutyric acid (GABA) can exert a trophic influence during development, expanding the role of this amino acid beyond its function as an inhibitory neurotransmitter. Proliferating precursor cells in the neocortical ventricular zone (VZ) express functional GABA(A) receptors as do immature postmigratory neurons in the developing cortical plate (CP); however, GABA(A) receptor properties in these distinct cell populations have not been compared. Using electrophysiological techniques in embryonic and early postnatal neocortex, we find that GABA(A) receptors expressed by VZ cells have a higher apparent affinity for GABA and are relatively insensitive to receptor desensitization compared with neurons in the CP. GABA-induced current magnitude increases with maturation with the smallest responses found in recordings from precursor cells in the VZ. No evidence was found that GABA(A) receptors on VZ cells are activated synaptically, consistent with previous data suggesting that these receptors are activated in a paracrine fashion by nonsynaptically released ligand. After neurons are born and migrate to the CP, they begin to demonstrate spontaneous synaptic activity, the majority of which is GABA(A) mediated. These spontaneous GABA(A) postsynaptic currents (sPSCs) first were detected at embryonic day 18 (E18). At birth, approximately 50% of recordings from cortical neurons demonstrated GABA(A)-mediated sPSCs, and this value increased with age. GABA(A)-mediated sPSCs were action potential dependent and arose from local GABAergic interneurons. GABA application could evoke action potential-dependent PSCs in neonatal cortical neurons, suggesting that during the first few postnatal days, GABA can act as an excitatory neurotransmitter. Finally, N-methyl-D-aspartate (NMDA)- but not non-NMDA-mediated sPSCs were also present in early postnatal neurons. These events were not observed in cells voltage clamped at negative holding potentials (-60 to -70 mV) but were evident when the holding potential was set at positive values (+30 to +60 mV). Together these results provide evidence for the early maturation of GABAergic communication in the neocortex and a functional change in GABA(A)-receptor properties between precursor cells and early postmitotic neurons. The change in GABA(A)-receptor properties may reflect the shift from paracrine to synaptic receptor activation.

A convenient electrode holder for glass pipettes to stabilize electrode potentials.

Electrophysiological techniques require stable, reversible electrodes for accurate recording. Ag/AgCl electrodes are commonly used in patch-clamp and intracellular recording pipettes, but when these electrodes are exposed to Cl--free media they are no longer reversible and may develop large, unstable offset potentials. We present here a simple electrode holder design that provides a liquid junction between the electrode solution and a KCl-filled reservoir containing the AgCl pellet. This allows an electrode solution of any composition to be used without affecting the reversibility of the electrode, and the liquid junction potentials that develop are small and well defined. Mechanical isolation of the AgCl pellet from the pipette improves long-term stability of the electrode because the AgCl coating is not scraped while changing pipettes. The response time of the holder and its wide-band noise levels are comparable to those of standard holders. For practical experiment durations, the diffusion for KCl from the reservoir down the pipette produces negligible contamination at the tip. Similarly, back diffusion of the pipette contents into the reservoir is also negligible.

Patterns of intracellular calcium fluctuation in precursor cells of the neocortical ventricular zone.

Changes in intracellular free calcium concentration ([Ca2+]i) are known to influence a variety of events in developing neurons. Although spontaneous changes of [Ca2+]i have been examined in immature cortical neurons, the calcium dynamics of cortical precursor cells have received less attention. Using an intact cortical mantle and confocal laser microscopy, we examined the spatiotemporal patterns of spontaneous [Ca2+]i fluctuations in neocortical ventricular zone (VZ) cells in situ. The majority of activity consisted of single cells that displayed independent [Ca2+]i fluctuations. These events occurred in cells throughout the depth of the VZ. Immunohistochemical staining confirmed that these events occurred primarily in precursor cells rather than in postmitotic neurons. When imaging near the ventricular surface, synchronous spontaneous [Ca2+]i increases were frequently observed in pairs of adjacent cells. Cellular morphology, time-lapse imaging, and nuclear staining demonstrated that this activity occurred in mitotically active cells. A third and infrequently encountered pattern of activity consisted of coordinated spontaneous increases in [Ca2+]i in groups of neighboring VZ cells. The morphological characteristics of these cells and immunohistochemical staining suggested that the coordinated events occurred in gap junction-coupled precursor cells. All three patterns of activity were dependent on the release of Ca2+ from intracellular stores. These results demonstrate distinct patterns of spontaneous [Ca2+]i change in cortical precursor cells and raise the possibility that these dynamics may contribute to the regulation of neurogenesis.

Nonsynaptic glycine receptor activation during early neocortical development.

Glycine receptors (GlyRs) contribute to fast inhibitory synaptic transmission in the brain stem and spinal cord. GlyR subunits are expressed in the developing neocortex, but a neurotransmitter system involving cortical GlyRs has yet to be demonstrated. Here, we show that GlyRs in immature neocortex are excitatory and activated by a nonsynaptically released endogenous ligand. Of the potential ligands for cortical GlyRs, taurine is by far the most abundant in the developing neocortex. We found that taurine is stored in immature cortical neurons and that manipulations known to elevate extracellular taurine cause GlyR activation. These data indicate that nonsynaptically released taurine activates GlyRs during neocortical development. As fetal taurine deprivation can cause cortical dysgenesis, it is possible that taurine influences neocortical development by activating GlyRs.

Postnatal development of low [Mg2+] oscillations in neocortex.

One form of rhythmic activity intrinsic to neocortex can be induced in slices of adult somatosensory cortex by lowering [Mg2+]o to unblock N-methyl--aspartate (NMDA) receptors. It has been suggested that a population of intrinsically burst-firing (IB) neurons that are unique to cortical layer 5 may play a role in the rhythmic activity seen under these conditions. Whole cell patch-clamp and field-potential recordings in slices of somatosensory cortex from neonatal rats were used to study the development of IB cells and the development of 0 [Mg2+] oscillations. IB cells were not encountered before postnatal day 12 (P12) in layer 5, but from P13 to P19 an increasing proportion of cells had IB properties. Recordings from cells at P7, P17, and P19 in 0 [Mg2+] indicate that dramatic changes occur postnatally in 0 [Mg2+]-induced activity. At P7, cells largely showed trains of single action potentials. In contrast, at P19, cells showed organized bursts of rhythmic activity lasting 0.5-5 s separated by periods of relative quiescence. Cells recorded at P17 were found to have less organized rhythmic activity than cells from P19 cortex. Field-potential recordings in 0 [Mg2+] made at P7 showed infrequent and slowly occurring field depolarizations, whereas field-potential recordings at P19 consisted of spontaneous bursts of 4-12 Hz oscillations identical to those observed in the adult. Application of NE, which inhibits burst-firing of layer 5 IB cells, significantly altered the discharge pattern of 0 [Mg2+] oscillations at P19. These data suggest that the maturation of one type of rhythmic network activity intrinsic to neocortex is influenced by the development of the membrane properties of a single cell type.

Cell coupling and uncoupling in the ventricular zone of developing neocortex.

Cells within the ventricular zone (VZ) of developing neocortex are coupled together into clusters by gap junction channels. The specific role of clustering in cortical neurogenesis is unknown; however, clustering provides a means for spatially restricted local interactions between subsets of precursors and other cells within the VZ. In the present study, we have used a combination of 5-bromo-2'-deoxyuridine (BrDU) pulse labeling, intracellular biocytin labeling, and immunocytochemistry to determine when in the cell cycle VZ cells couple and uncouple from clusters and to determine what cell types within the VZ are coupled to clusters. Our results indicate that clusters contain radial glia and neural precursors but do not contain differentiating or migrating neurons. In early neurogenesis, all precursors in S and G2 phases of the cell cycle are coupled, and approximately half of the cells in G1 are coupled. In late neurogenesis, however, over half of the cells in both G1 and S phases are not coupled to VZ clusters, whereas all cells in G2 are coupled to clusters. Increased uncoupling in S phase during late neurogenesis may contribute to the greater percentage of VZ cells exiting the cell cycle at this time. Consistent with this hypothesis, we found that pharmacologically uncoupling VZ cells with octanol decreases the percentage of VZ cells that enter S phase. These results demonstrate that cell clustering in the VZ is restricted to neural precursors and radial glia, is dynamic through the cell cycle, and may play a role in regulating neurogenesis.

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex.

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.

Mechanisms underlying neuronal migration disorders and epilepsy.

Neuronal migration disorders are often associated with intractable epilepsy. These cortical malformations are quite heterogeneous, suggesting that they may result from interference with a diverse set of processes during corticogenesis. Progress toward understanding the pathophysiologic basis of these disorders is coming from research into the basic mechanisms of corticogenesis, animal models of cortical malformations, and molecular genetic approaches to migration disorders.