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Axonal and dendritic endocytic pathways in cultured neurons.

The endocytic pathways from the axonal and dendritic surfaces of cultured polarized hippocampal neurons were examined. The dendrites and cell body contained extensive networks of tubular early endosomes which received endocytosed markers from the somatodendritic domain. In axons early endosomes were confined to presynaptic terminals and to varicosities. The somatodendritic but not the presynaptic early endosomes were labeled by internalized transferrin. In contrast to early endosomes, late endosomes and lysosomes were shown to be predominantly located in the cell body. Video microscopy was used to follow the transport of internalized markers from the periphery of axons and dendrites back to the cell body. Labeled structures in both domains moved unidirectionally by retrograde fast transport. Axonally transported organelles were sectioned for EM after video microscopic observation and shown to be large multivesicular body-like structures. Similar structures accumulated at the distal side of an axonal lesion. Multivesicular bodies therefore appear to be the major structures mediating transport of endocytosed markers between the nerve terminals and the cell body. Late endocytic structures were also shown to be highly mobile and were observed moving within the cell body and proximal dendritic segments. The results show that the organization of the endosomes differs in the axons and dendrites of cultured rat hippocampal neurons and that the different compartments or stages of the endocytic pathways can be resolved spatially.

Animals↗

Cell-type-specific expression of catecholamine transporters in the rat brain.

The dopamine transporter (DAT) and norepinephrine transporter (NET) terminate catecholaminergic neurotransmission at synapses by high-affinity sodium-dependent reuptake into presynaptic terminals, and are the initial sites of action for drugs of abuse and antidepressants. In the present study, we used in situ hybridization combined with immunohistochemistry to study the distribution of DAT and NET mRNA in the adult rat brain. Cells were first immunolabeled with antisera directed against one of the catecholamine-synthetic enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), or phenylethanolamine-N-methyltransferase (PNMT), in order to identify dopaminergic, noradrenergic, or epinephrine-containing cells. The immunolabeled cells were subsequently assayed for their ability to express catecholamine transporter mRNAs by in situ hybridization using either a rat DAT or NET cRNA probe. All dopaminergic cell groups of the mesencephalon contained high levels of DAT mRNA but only the A12 and A13 dopaminergic cell groups of the diencephalon appear to express detectable levels of DAT. All norepinephrine-containing cell bodies in the brainstem (locus coeruleus and lateral tegmentum) appear to express NET mRNA. In contrast, epinephrine-containing cell bodies of the brainstem do not appear to express NET mRNA, which raises the possibility that epinephrine may utilize a transporter that is distinct from the other bioactive amines, or may act as an endocrine regulator that does not require rapid reuptake mechanisms. Moreover, the cell-type-specific expression of catecholamine transporters suggests that DAT and NET gene expression may be closely linked to cellular mechanisms that specify transmitter phenotype. The termination of neurotransmission is a critical component of neural signaling and depends on the rapid removal of neurotransmitters from the synaptic cleft. Pharmacological evidence indicates that the action of monoamines at the synapse is terminated predominantly by rapid reuptake into presynaptic nerve endings via neurotransmitter-specific, high-affinity, Na(+)-dependent membrane transporter proteins. The cDNAs encoding distinct transporter proteins for the monoamines dopamine, norepinephrine, and serotonin have been cloned, expressed, and characterized in a variety of heterologous systems (Blakely et al., 1991; Giros et al., 1991; Hoffman et al., 1991; Kilty et al., 1991; Pacholczyk et al., 1991; Shimada et al., 1991; Usdin et al., 1991). Although the monoamine transporters share a high degree of sequence homology, they are distinguished by their monoamine substrate specificities and by their differential sensitivities to a wide spectrum of transport antagonists. For example, pharmacological agents that potently inhibit norepinephrine and serotonin transport, such as desmethylimipramine and citalopram, have little effect on the activity of the dopamine transporter (Javitch et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Neuronal excitability: voltage-dependent currents and synaptic transmission.

Neuronal membrane excitability and the synaptic connections among neurons produce behavior and cognition. The intracellular compartment of neurons is negatively charged relative to the extracellular space, and this charge, as well as current flow, is produced by ions. From the perspective of charged ions, the lipid bilayer of the neuronal membrane acts as a capacitor, and transmembrane glycoprotein pores or channels act as resistors. The open and closed states of ionic channels determine the membrane potential. At equilibrium, the lowest resistance or greatest permeability is for potassium, and the resting membrane potential is close to the equilibrium potential for potassium. When a channel is opened, permeable ions diffuse down their electrochemical gradients and the membrane potential is changed. Channels are gated (opened or closed) by voltage, neurotransmitters, and second messengers. The neuron integrates synaptic potentials produced by transmitter-gated channel activity and either generates a subthreshold potential, or a suprathreshold depolarization that generates an action potential or a burst of action potentials. Action potential generation is mediated by a large, brief sodium influx that is followed by activation of a voltage-dependent potassium eflux. The pattern of action potential firing is dependent on the interaction of a repertoire of voltage-dependent ion conductances. The action potential is the main signaling mechanism to activate synaptic transmission at axon terminals. Synaptic transmission is graded depending on the amount of calcium entering the presynaptic terminal. The number of action potentials, or the shape of the action potential, will determine the amount of calcium entering the terminal and the efficacy of synaptic transmission. Presynaptic ion channels may also be controlled by neurotransmitters or modulators and affect synaptic transmission by altering the amount of calcium influx.

Animals↗

Can presynaptic depolarization release transmitter without calcium influx?

Recent experimental evidence suggesting that presynaptic depolarization can evoke transmitter release without calcium influx has been re-examined. The presynaptic terminal of the squid giant synapse can be depolarized by variable amounts while recording presynaptic calcium current under voltage clamp and postsynaptic responses. Small depolarizations open few calcium channels with large single channel currents. Large depolarizations approaching the calcium equilibrium potential open many channels with small single channel currents. When responses to small and large depolarizations eliciting similar total macroscopic calcium currents are compared, the large pulses evoke more transmitter release. This apparent voltage-dependence of transmitter release may be explained by the greater overlap of calcium concentration domains surrounding single open calcium channels when many closely apposed channels open at large depolarizations. This channel domain overlap leads to higher calcium concentrations at transmitter release sites and more release for large depolarizations than for small depolarizations which open few widely dispersed channels. At neuromuscular junctions, a subthreshold depolarizing pulse to motor nerve terminals may release over a thousand times as much transmitter if it follows a brief train of presynaptic action potentials than if it occurs in isolation. This huge synaptic facilitation has been taken as indicative of a direct effect of voltage which is manifest only when prior activity raises presynaptic resting calcium levels. This large facilitation is actually due to a post-tetanic supernormal excitability in motor nerve terminals, causing the previously subthreshold test pulse to become suprathreshold and elicit a presynaptic action potential. When motor nerve terminals are depolarized by two pulses, as the first pulse increases above a certain level it evokes more transmitter release but less facilitation of the response to the second pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Anatomical distribution and ultrastructural organization of the GABAergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies.

gamma-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I-III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I-III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons. At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals. These results confirm data obtained with L-glutamate decarboxylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.

Animals↗

Evidence for activity-dependent modulation of sensory-terminal excitability in spindles by glutamate release from synaptic-like vesicles.

Sensory terminals of muscle spindles and similar mechanosensory neurons contain large numbers of 50 nm, "synaptic-like" vesicles (SLVs), about whose role very little is known. Using fluorescence microscopy, immunocytochemistry and electrophysiological recording, we present evidence that SLVs undergo a recycling process, and that they release glutamate that has an autogenic excitatory effect on mechanosensory transduction, probably involving a metabotropic receptor linked to phospholipase D. The rate of recycling of SLVs is activity dependent, at least in part, as shown by an increased rate of destaining of preparations labelled with FMI-43 during high-frequency, small-amplitude vibration. Immunogold labelling showed levels of glutamate-like reactivity in the sensory terminals at least as great as in probable Ia presynaptic terminals in the spinal cord. Exogenously applied glutamate has an excitatory effect on the spindle's response to stretch, which is blocked by 3,5-dihydroxyphenylglycine.

Animals↗

Effects of GABA on presumed presynaptic Ca2+ entry in hippocampal slices.

Evoked field potentials and changes in [Ca2+]o were measured in the 'in vitro' hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/commissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca2+]o. In contrast to findings in normal medium these decreases in [Ca2+]o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca2+ in low Ca2+ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca2+ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.

Animals↗

Persistent enhancement of transmitter release accompanying long-term potentiation in the guinea pig hippocampus.

In order to examine temporal changes in enhancement of transmitter release during long-term potentiation (LTP), we examined amplitude fluctuation of excitatory postsynaptic potentials (EPSPs) for longer periods than 2 h after tetanic stimulation (up to 4 h in the longest observation). The relative magnitude of excitatory postsynaptic potentiation (EPSP) fluctuation (coefficient of variation, CV) reduced throughout the observation periods in association with an increase in EPSP amplitude after tetanic stimulation. The reciprocals of squared CVs (= mean2/variance) were almost in proportion to the magnitude of LTP, and the ratio of 1/CV2 and the LTP magnitude did not change significantly for up to 4 h. These findings suggest that a prolonged enhancement of transmitter release from presynaptic terminals underlies LTP, and the relative contribution of this presynaptic enhancement does not change significantly for 2 h (maybe up to 4 h, or longer) after tetanic stimulation.

Animals↗

Impairment of syntaxin by botulinum neurotoxin C1 or antibodies inhibits acetylcholine release but not Ca2+ channel activity.

The involvement of syntaxin, an omega-conotoxin-sensitive Ca2+ channel-associated protein, in acetylcholine release was studied at synapses formed between rat sympathetic neurons in culture. Transmission at these synapses involved omega-conotoxin-sensitive Ca2+ channels because a dose-dependent inhibition was observed when omega-conotoxin was bath-applied. Confocal microscope examination of immunofluorescent staining showed that syntaxin had a similar distribution to synaptic vesicle-associated membrane proteins, synaptophysin and vesicle-associated membrane protein/synaptobrevin-2, indicating that syntaxin molecules are concentrated in the presynaptic terminals. Botulinum neurotoxin C1 applied extracellularly or intracellularly into presynaptic neurons blocked synaptic transmission. Introduction of a monoclonal antibody, or polyclonal antibodies, to syntaxin into the presynaptic neuron depressed the evoked release of acetylcholine without affecting Ca2+ influx through Ca2+ channels. These results suggest that syntaxin plays an important role in release of neurotransmitter by a nerve impulse and that this mechanism is downstream of Ca2+ influx.

Acetylcholine↗

Ultrastructural localization of GABA-immunoreactive terminals in the anteroventral cochlear nucleus of the guinea pig.

The immunocytochemical distribution of gamma-aminobutyric acid (GABA) was studied by electron microscopy in the anteroventral cochlear nucleus (AVCN) of the guinea pig using affinity-purified antibodies made against GABA conjugated to bovine serum albumin. Our observations confirm that spherical cells are the predominant cell type in the guinea pig AVCN and receive numerous axosomatic contacts (Schwartz and Gulley, (1978) J. Anat. 153, 489-508). Stellate cells receive few axosomatic contacts. Electron microscopic immunocytochemistry shows that GABA immunoreactivity is present in synaptic terminals in the AVCN. Of the several classes of presynaptic terminals present in the AVCN as characterized by vesicle type (large round; oval/pleomorphic; flat; small round) only those containing oval/pleomorphic vesicles were GABA-immunoreactive. However, GABA immunoreactivity may not be present in all these terminals because some oval/pleomorphic terminals are unlabelled. Immunoreactive terminals are widespread in the AVCN; they are abundant on spherical cell bodies, rarely seen on stellate cell bodies and are also found scattered throughout the neuropile.

Animals↗

High-frequency firing helps replenish the readily releasable pool of synaptic vesicles.

Synapses in the central nervous system undergo various short- and long-term changes in their strength, but it is often difficult to distinguish whether presynaptic or postsynaptic mechanisms are responsible for these changes. Using patch-clamp recording from giant synapses in the mouse auditory brainstem, we show here that short-term synaptic depression can be largely attributed to rapid depletion of a readily releasable pool of vesicles. Replenishment of this pool is highly dependent on the recent history of synaptic activity. High-frequency stimulation of presynaptic terminals significantly enhances the rate of replenishment. Broadening the presynaptic action potential with the potassium-channel blocker tetraethylammonium, which increases Ca2+ entry, further enhances the rate of replenishment. As this increase can be suppressed by the Ca2+-channel blocker Cd2+ or by the Ca2+ buffer EGTA, we conclude that Ca2+ influx through voltage-gated Ca2+ channels is the key signal that dynamically regulates the refilling of the releasable pool of synaptic vesicles in response to different patterns of inputs.

Action Potentials↗

The growth cones of Aplysia sensory neurons: Modulation by serotonin of action potential duration and single potassium channel currents.

Serotonin (5-HT) closes a specific K channel ("S") in the cell body of Aplysia sensory neurons, resulting in a slow excitatory postsynaptic potential and spike broadening. To determine whether the S channel is present and can be modulated in processes of the neuron other than the cell body, we studied the effects of 5-HT on growth cones of sensory neurons in culture by using the patch-clamp technique. Simultaneous application of 5-HT to the cell body and to the growth cones of sensory neurons produced, in both, a slow depolarization of approximately 5 mV. Also, 5-HT produced a lengthening of the duration of action potential in the growth cone and cell body by 20-30%. Similar effects were observed in isolated growth cones that had been severed from the rest of the neuron, implying that the growth cones contain all the molecular components (i.e., receptors, channels, cAMP cascade) necessary for 5-HT action. Cell-attached patch-clamp recordings demonstrated the presence of S channels in sensory neuron growth cones. Application of serotonin to the bath produced long-lasting all-or-none closures of these channels in a manner identical to the previously characterized action of 5-HT in the cell body. Thus, channel modulation is not restricted to the cell body and probably occurs throughout the sensory neuron. This strengthens the view that S-channel modulation may also occur at the sensory neuron presynaptic terminal, where it could play a role in the presynaptic facilitation produced by 5-HT.

Journal Article↗

Methylazoxymethanol-induced aberrant Purkinje cell dendritic development.

Purkinje cell dendrites develop with a specific orientation and relationship to related neurons and glia. Their dendritic spine postsynaptic membrane specialization may, in turn, require a permanent presynaptic contact by parallel fibers. To determine whether changes in the surrounding cells influence the normal development of the cerebellar Purkinje cell dendrites and spine specializations, destruction on the differentiating cell layer was induced in the postnatal mouse by administration of methylazoxymethanol acetate (MAM) (0.05 micronl/gm body weight) at day zero. The Purkinje cells were examined by light and electron microscopy on the tenth postnatal day. The midsagittal surface area of the cerebellar vermis in treated animals was reduced by an average of 60%. MAM-induced granule cell depletion and Purkinje cell dislocation were observed by light microscopy. When compared to controls, examination following Golgi impregnation revealed random orientation of Purkinje cell apical poles, and multiple primary dendrites of reduced length with few branches, branchlets and spines. Vertical processes of Golgi epithelial (Bergmann) cells were obliquely directed, reduced in length and complexity in MAM-treated mice. Ultrastructural examination revealed naked Purkinje cell dendritic spine specializations in both groups. Although necrotic debris persisted in astrocytes and macrophages, degenerating presynaptic terminals were not found. This study suggests that permanent presynaptic contact by parallel fibers is not essential for spine development. Astrocytic reactions to injury, in association with the reduced folial expansion, may have contributed to the observed abnormalities and disorientation of the Purkinje cells. The data suggests that Purkinje cell dendritic development may be strongly influenced by changes in surrounding cells.

Animals↗

GABAergic synapses in the antennal lobe and mushroom body of the locust olfactory system.

To help elucidate the role of inhibitory feedback in the genesis of odour-evoked synchronization of neural activity, we investigated the distribution of gamma-aminobutyric acid (GABA)ergic synaptic terminals in the antennal lobes (AL) and mushroom bodies (MB) of the locust olfactory system. Electron-microscopy, intracellular horseradish peroxidase labelling, and immunocytochemistry were combined to assess the distribution of GABAergic synapses, using established methods (Leitch and Laurent [1993] J. Comp. Neurol. 337:461-470). In the AL, GABA-immunoreactive presynaptic terminals contacted both immunoreactive and immunonegative profiles. Conversely, GABA-immunoreactive profiles received direct input from both reactive and negative terminals. The tract containing the axons of the projection neurons that run from the AL to the MB contained about 830 axons of fairly uniform size, none of which was immunoreactive for GABA. In the calyx of the MB, large immunoreactive terminals contacted very-small-diameter profiles thought to belong to the Kenyon cells (KCs). This was confirmed by combining immunocytochemistry with intracellular HRP-labelling of KCs. KCs were not immunoreactive for GABA. Although some GABAergic contacts were made onto the spiny profiles of KCs, others were made onto their dendritic shafts. Large GABA-immunoreactive profiles were also found to contact large negative profiles that were presynaptic to KC terminals. This suggests that KC dendrites can be both pre- and post-synaptically inhibited in the calyx. The MB pedunculus contained ca. 50,000 tightly packed KC axons, showing conspicuous en passant and often reciprocal synaptic contacts between neighbouring axons. KC axons were immunonegative, but received direct input from, and contacted directly, large immunoreactive profiles running across or along the KC axons. In the alpha- and beta-lobes of the MB, connections similar to those in the pedunculus were seen with two main differences: (1) The density of synaptic profiles was higher, giving on occasion numerous serially connected profiles in a single section; (2) large immunonegative profiles with dense-core vesicles were abundant and were frequently presynaptic to GABAergic processes and to very-small-diameter profiles which possibly belong to KCs. These results are discussed in the context of the known physiological data on olfactory processing in these complex circuits.

Animals↗

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Animals↗

Crayfish motor nerve terminal's response to serotonin examined by intracellular microelectrode.

Measurements of resting potential and action potential in presynaptic branches of the excitatory motor axon to the crayfish opener muscle were made with intracellular microelectrodes during application of serotonin (10(-9)-10(-3) M). A 5-min exposure to 10(-6) M serotonin produced enhancement of excitatory junction potentials (EJPs) lasting about 1 h. The membrane potential of the presynaptic terminal was depolarized by about 5 mV; the depolarization subsided within 1/2 h. Concomitant reduction in amplitude of the presynaptic action potential, not accompanied by spike broadening, was observed. The presynaptic depolarization, and the enhancement of EJPs, were dependent on the presence of extracellular sodium but not extracellular calcium. A possible mechanism for serotonin's effect involves initial entry of sodium into the nerve terminal, with consequent increased availability of intracellular calcium. The subsequent long-lasting phase of EJP enhancement may result from an additional effect on the metabolism of the nerve terminal.

Action Potentials↗

Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release.

Injections of inositol trisphosphate (IP3) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysia californica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca2+ release from various Ca2+ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP3-induced calcium release with heparin, abolished the effects of IP3, but not the effects of NAADP, suggesting the presence of an intracellular Ca2+ pool independent of those containing ryanodine receptors (RyR) or IP3 receptors. To reinforce electrophysiological observations, intracellular [Ca2+]i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca2+ concentration. RyR- and IP3-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP3-dependent, and NAADP-dependent Ca2+ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca2+ concentration that triggers transmitter release.

Acetylcholine↗

Increased dopamine uptake in striatal synaptosomes after treatment of rats with amantadine.

The aim of the present study was to investigate the effect of short- and long-term treatments with amantadine on the activity of the neuronal dopamine transporter (DAT) in the rat striatum. For this purpose, the [3H]dopamine uptake was measured in striatal synaptosomes prepared from rats treated for 2, 7 and 14 days with amantadine (40 mg/kg; i.p.). After 7 days of treatment, amantadine increased the apparent V(max) by 30% without modification of the apparent K(m) of dopamine uptake whereas no change in these parameters was observed after 2 and 14 days treatment. Binding assays conducted with [3H]GBR-12935 on membranes prepared from animals treated with amantadine revealed no difference in the density and the affinity of striatal DAT binding sites as compared to control. This indicates that the increased dopamine uptake was not reflecting a modification at the level of the DAT expression. The activity of the DAT is regulated by phosphorylation and one may propose that ionotropic glutamate receptors present on presynaptic terminals directly modulate this phosphorylation. An indirect mechanism would involve presynaptic dopamine receptors that control the activity of the DAT in response to the increased dopamine concentration in the synaptic cleft.

Amantadine↗