Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.

Quantal acetylcholine release induced by mediatophore transfection.

Mediatophore is a protein of approximately 200 kDa able to translocate acetylcholine in response to calcium. It was purified from the presynaptic plasma membranes of the electric organ nerve terminals. Mediatophore is a homooligomer of a 16-kDa subunit, homologous to the proteolipid of V-ATPase. Cells of the N18TG-2 neuronal line are not able to produce quantal acetylcholine release. We show here that transfection of N18TG-2 cells with a plasmid encoding the mediatophore subunit restored calcium-dependent release. The essential feature of such a release was its quantal nature, similar to what is observed in situ in cholinergic synapses from which mediatophore was purified.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream consequences of an excitotoxic stimulus; (ii) palmitoylethanolamide, unlike anandamide, behaves as an endogenous agonist for CB2-like receptors on granule cells; and (iii) activation of such receptors may serve to downmodulate deleterious cellular processes following pathological events or noxious stimuli in both the nervous and immune systems.

N-methyl-D-aspartate receptor-induced proteolytic conversion of postsynaptic class C L-type calcium channels in hippocampal neurons.

Ca2+ influx controls multiple neuronal functions including neurotransmitter release, protein phosphorylation, gene expression, and synaptic plasticity. Brain L-type Ca2+ channels, which contain either alpha 1C or alpha 1D as their pore-forming subunits, are an important source of calcium entry into neurons. Alpha 1C exists in long and short forms, which are differentially phosphorylated, and C-terminal truncation of alpha 1C increases its activity approximately 4-fold in heterologous expression systems. Although most L-type calcium channels in brain are localized in the cell body and proximal dendrites, alpha 1C subunits in the hippocampus are also present in clusters along the dendrites of neurons. Examination by electron microscopy shows that these clusters of alpha 1C are localized in the postsynaptic membrane of excitatory synapses, which are known to contain glutamate receptors. Activation of N-methyl-D-aspartate (NMDA)-specific glutamate receptors induced the conversion of the long form of alpha 1C into the short form by proteolytic removal of the C terminus. Other classes of Ca2+ channel alpha1 subunits were unaffected. This proteolytic processing reaction required extracellular calcium and was blocked by inhibitors of the calcium-activated protease calpain, indicating that calcium entry through NMDA receptors activated proteolysis of alpha1C by calpain. Purified calpain catalyzed conversion of the long form of immunopurified alpha 1C to the short form in vitro, consistent with the hypothesis that calpain is responsible for processing of alpha 1C in hippocampal neurons. Our results suggest that NMDA receptor-induced processing of the postsynaptic class C L-type Ca2+ channel may persistently increase Ca2+ influx following intense synaptic activity and may influence Ca2+-dependent processes such as protein phosphorylation, synaptic plasticity, and gene expression.

The resource consumption principle: attention and memory in volumes of neural tissue.

In the cerebral cortex, the small volume of the extracellular space in relation to the volume enclosed by synapses suggests an important functional role for this relationship. It is well known that there are atoms and molecules in the extracellular space that are absolutely necessary for synapses to function (e.g., calcium). I propose here the hypothesis that the rapid shift of these atoms and molecules from extracellular to intrasynaptic compartments represents the consumption of a shared, limited resource available to local volumes of neural tissue. Such consumption results in a dramatic competition among synapses for resources necessary for their function. In this paper, I explore a theory in which this resource consumption plays a critical role in the way local volumes of neural tissue operate. On short time scales, this principle of resource consumption permits a tissue volume to choose those synapses that function in a particular context and thereby helps to integrate the many neural signals that impinge on a tissue volume at any given moment. On longer time scales, the same principle aids in the stable storage and recall of information. The theory provides one framework for understanding how cerebral cortical tissue volumes integrate, attend to, store, and recall information. In this account, the capacity of neural tissue to attend to stimuli is intimately tied to the way tissue volumes are organized at fine spatial scales.

Short-term synaptic enhancement and long-term potentiation in neocortex.

Repetitive stimuli reliably induce long-term potentiation (LTP) of synapses in the upper layers of the granular somatosensory cortex but not the agranular motor cortex of rats. Herein we examine, in these same cortical areas, short-term changes in synaptic strength that occur during the LTP induction period. theta-Burst stimulation produced a strong short-term enhancement of synapses in the granular area but only weak enhancement in the agranular area. The magnitude of enhancement during stimulation was strongly correlated with the magnitude of LTP subsequently expressed. Short-term enhancement was abolished by an antagonist of N-methyl-D-aspartate (NMDA) receptors but remained in the presence of a non-NMDA receptor antagonist. Inhibitory postsynaptic potentials of the granular and agranular areas displayed similar frequency sensitivity, but the frequency sensitivity of NMDA receptor-dependent excitatory postsynaptic potentials differed significantly between areas. We propose that pathway-specific differences in short-term enhancement are due to variations in the frequency dependence of NMDA currents; different capacities for short-term enhancement may explain why repetitive stimulation more readily induces LTP in the somatosensory cortex than in the motor cortex.

Emergence of order in visual system development.

Neural connections in the adult central nervous system are highly precise. In the visual system, retinal ganglion cells send their axons to target neurons in the lateral geniculate nucleus (LGN) in such a way that axons originating from the two eyes terminate in adjacent but nonoverlapping eye-specific layers. During development, however, inputs from the two eyes are intermixed, and the adult pattern emerges gradually as axons from the two eyes sort out to form the layers. Experiments indicate that the sorting-out process, even though it occurs in utero in higher mammals and always before vision, requires retinal ganglion cell signaling; blocking retinal ganglion cell action potentials with tetrodotoxin prevents the formation of the layers. These action potentials are endogenously generated by the ganglion cells, which fire spontaneously and synchronously with each other, generating "waves" of activity that travel across the retina. Calcium imaging of the retina shows that the ganglion cells undergo correlated calcium bursting to generate the waves and that amacrine cells also participate in the correlated activity patterns. Physiological recordings from LGN neurons in vitro indicate that the quasiperiodic activity generated by the retinal ganglion cells is transmitted across the synapse between ganglion cells to drive target LGN neurons. These observations suggest that (i) a neural circuit within the immature retina is responsible for generating specific spatiotemporal patterns of neural activity; (ii) spontaneous activity generated in the retina is propagated across central synapses; and (iii) even before the photoreceptors are present, nerve cell function is essential for correct wiring of the visual system during early development. Since spontaneously generated activity is known to be present elsewhere in the developing CNS, this process of activity-dependent wiring could be used throughout the nervous system to help refine early sets of neural connections into their highly precise adult patterns.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

Impairment of axonal development and of synaptogenesis in hippocampal neurons of synapsin I-deficient mice.

Synapsin I, the most abundant of all neuronal phosphoproteins, is enriched in synaptic vesicles. It has been hypothesized to regulate synaptogenesis and neurotransmitter release from adult nerve terminals. The evidence for such roles has been highly suggestive but not compelling. To evaluate the possible involvement of synapsin I in synaptogenesis and in the function of adult synapses, we have generated synapsin I-deficient mice by homologous recombination. We report herein that outgrowth of predendritic neurites and of axons was severely retarded in the hippocampal neurons of embryonic synapsin I mutant mice. Furthermore, synapse formation was significantly delayed in these mutant neurons. These results indicate that synapsin I plays a role in regulation of axonogenesis and synaptogenesis.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Molecular cloning and functional characterization of the Xenopus Ca(2+)-binding protein frequenin.

Frequenin was originally identified in Drosophila melanogaster as a Ca(2+)-binding protein facilitating transmitter release at the neuromuscular junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The deduced protein has 70% identity with Drosophila frequenin and about 38-58% identity with other Ca(2+)-binding proteins. The most prominent features are the four EF-hands, Ca(2+)-binding motifs. Xfreq mRNA is abundant in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adult brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well with the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant protein was introduced into Xenopus embryonic spinal neurons by early blastomere injection. Synapses made by spinal neurons containing exogenous Xfreq exhibited a much higher synaptic efficacy. These results provide direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in synaptic development and plasticity.

The kinetics of quantal transmitter release from retinal amacrine cells.

Exocytosis of transmitter at most synapses is a very fast process triggered by the entry of Ca2+ during an action potential. A reasonable expectation is that the fast step of exocytosis is followed by slow steps readying another vesicle for exocytosis but the identity and kinetics of these steps are presently unclear. By voltage clamping both pre- and postsynaptic neurons in an isolated pair of retinal amacrine cells, we have measured evoked synaptic currents and responses to single vesicles of transmitter (minis). From these currents, we have computed the rate of exocytosis during a sustained presynaptic depolarization. We show here that for these cells, release is consistent with a scheme of "fire and reload." Large Ca2+ influx causes the rapid release of a small number of vesicles, typically approximately 10 per presynaptic neuron, likely corresponding to those vesicles already docked. After this spike of exocytosis whose peak is 150 quanta per release site per s, continued Ca2+ influx sustains release at only 22 quanta per release site per s, probably rate-limited by the docking of fresh vesicles.

Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.