Pre- and post-synaptic structures in insect CNS: intramembranous features and sites of alpha-bungarotoxin binding

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous articles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated alpha-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that alpha-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.     

Rhombic particle arrays in gill epithelium of a mollusc, Aplysia californica.

Gill epithelia from adult and juvenile Aplysia were examined by conventional thin section and freeze-fracture methods. Freeze-fracture replicas of adult gill epithelium revealed septate and gap junctions, which served as membrane markers for the epithelial cells. In these same cell membranes, non-junctional rhombic arrays of intramembranous particles were observed on prominent ridges on the membrane P fracture face of some epithelial cells. In thin sections of adult epithelium, nerve terminals were observed abutting the lateral plasma membranes near the basal lamina of some epithelial cells. Correlative areas of plasma membrane in freeze-fracture replicas showed a close association between rhombic particle arrays and abutting nerve terminals. In thin sections of juvenile Aplysia, nerve terminals abutting the epithelial cells were not recognizable, and rhombic arrays were not observed in freeze-fracture replicas. This suggested that a developmental association existed between the appearance of rhombic arrays in adult epithelia and their innervation. It is not known with certainty if, in invertebrates, rhombic arrays are an essential structural entity of all innervated cell membranes; however, in the cells thus far studied, there appears to be an associative condition. In the case of the gill epithelium of Aplysia, rhombic arrays are located in the same vicinity as the abutting nerve terminals. Similar arrays of intramembranous particles have been observed in myoneural postjunctional complexes of other invertebrates and have been interpreted to be the morphological expression of neurotransmitter receptors. An analogous explanation is put forth, namely that rhombic arrays may represent the structural correlates of neurotransmitter receptors and/or ionic channels in innervated membranes of invertebrates.     

Retrograde axon reaction following section of asynaptic nerve fibers

Summary The asynaptic spinal neurons of the gymnotid teleost Sternarchus albifrons show several distinct characteristics of the retrograde reaction of the perikaryon (which corresponds to chromatolysis in mammals) following axotomy. Nuclei of affected cells are characteristically eccentric. Large bundles of neurofilaments, never seen in normal perikarya of these cells, become prominent following axotomy. There is a marked increase in the number and size of dense bodies in the affected perikarya. Large arrays of parallel rough endoplasmic reticulum, never seen in normal cells, are frequent in the axotomized neurons. These results demonstrate that disconnection from synaptic terminals is not a necessary condition for the retrograde reaction of the perikaryon following axotomy.     

Pre- and post-synaptic structures in insect CNS: Intramembranous features and sites of α-bungarotoxin binding

The central neuropile of thoracic ganglia in the central nervous system (CNS) of the cockroach Periplaneta americana contains synapses with characteristic pre- and post-synaptic membrane specializations and associated structures. These include dense pre-synaptic T-bars surrounded by synaptic vesicles, together with post-synaptic densities of varying electron opacity. Exocytotic release of synaptic vesicles is observed only rarely near presynaptic densities, but coated pits are seen at variable distances from them, and may be involved in membrane retrieval. After freeze-fracture, paralinear arrays of intramembranous particles (IMPs) are detected on the P face of many presynaptic terminals, with associated dimples indicative of vesicular release. The E face of these membranes exhibits protuberances complementary to the P face dimples, as well as scattered larger IMPs. Post-synaptic membranes possess dense IMP aggregates on the P face, some of which may represent receptor molecules. Electrophysiological studies with biotinylated α-bungarotoxin reveal that biotinylation does not inhibit the pharmacological effectiveness of the toxin in blocking acetylcholine receptors on an identified motoneurone in the metathoracic ganglion. Preliminary thin section ultrastructural analysis of this tissue post-treated with avidin-HRP or avidin-ferritin indicates that α-bungarotoxin-binding sites are localized at certain synapses in these insect thoracic ganglia.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – I. Naturally occurring variability in active zone structure

The orderly arrays of intramembranous particles (IMPs) found in the p-face of freeze-fracture replicas of the frog neuromuscular junction (‘active zones’) are believed to be involved in transmitter release. Some or all of the particles represent voltage-dependent Ca2+ channels. Since there is a great heterogeneity in the amount of transmitter released by different frog motor nerve terminals we sought to determine whether active zone (AZ) structure displayed a similar heterogeneity by using a novel freeze-fracture procedure providing large, intact replicas containing significant portions of motor nerve terminals from the cutaneous pectoris muscle of the frog, Rana pipiens. Using only junctions in which more than 50 AZs or more than 50 μm of nerve terminal were included in the fractures, we measured AZ length, AZ intramembranous particle density, terminal width at each AZ, space between AZs, the angle of AZ orientation with respect to the longitudinal axis of the nerve terminal, exposed pre-synaptic nerve terminal surface area and a calculated value for mean AZ length per unit terminal length. The analysis led to the following conclusions. There is an approximate 5-fold range in mean AZ length/micrometre terminal length. Terminal width is a good predictor of AZ length. Particle density does not vary significantly within a given AZ, nor between AZs from the same or different junctions. The distance between AZs is not related to AZ length, i.e. shorter AZs are no more or less likely to be closer to the adjacent AZ. The probability of release from any AZ on action potential invasion is small. If most of the IMPs are Ca2+ channels, either the probability of channel opening or the efficacy of triggering release is very low or both. That the variability in release efficacy in different terminals is much greater than ultrastructural variability in terminals suggests that regulation of release is dominated by physiological processes that do not have obvious ultrastructural correlates. On the other hand, the apparent excess of AZ relative to the number of vesicles released indicates that the amount and variability in amount of AZ is important in ways that need to be elucidated.     

Sexual differentiation of identified motor terminals in Drosophila larvae

In Drosophila, we have found that some of the motor terminals in wandering third-instar larvae are sexually differentiated. In three out of the four body-wall muscle fibers that we examined, we found female terminals that produced a larger synaptic response than their male counterparts. The single motor terminal that innervates muscle fiber 5 produces an EPSP that is 69% larger in females than in males. This is due to greater release of transmitter from female than male synaptic terminals because the amplitude of spontaneous miniature EPSPs was similar in male and female muscle fibers. This sexual difference exists throughout the third-instar: it is seen in both early (foraging) and late (wandering) third-instar larvae. The sexual differentiation appears to be neuron specific and not muscle specific because the same axon produces Is terminals on muscle fibers 2 and 4, and both terminals produce larger EPSCs in females than males. Whereas, the Ib terminals innervating muscle fibers 2 and 4 are not sexually differentiated. The differences in transmitter release are not due to differences in the size of the motor terminals. For the terminal on muscle fiber 5 and the Is terminal on muscle fiber 4, there were no differences in terminal length, the number of branches, or the number of synaptic boutons in males compared to females. These sexual differences in neuromuscular synaptic physiology may be related to male-female differences in locomotion.     

Adenosine receptor mediated inhibition of noradrenaline release from slices of the rat hippocampus

Adenosine and adenosine analogues inhibited electrically evoked 3H-noradrenaline (3H-NA) release from slices of the rat hippocampus in vitro in a dose -dependent manner in the concentration range 0.01–100 M. L-phenylisopropyladenosine (L-PIA) was more potent than 5′-N-carboxamidoadenosine (NECA), which was more potent than adenosine. The adenosine uptake blocker dipyridamole (3 M) enhanced the effect of exogenous adenosine, and had a slight inhibitory effect per se. The effect of L-PIA on NA release was competitively antagonized by 8-phenyltheopylline; pA2=7.1. Enprophylline (300 M), theophylline (300 M) and 8-phenyltheophylline (1–10 M) enhanced the evoked 3H-NA release per se, while no such enhancement was seen with the non-xanthine phosphodiesterase inhibitor ZK 62.711 (Rolipram) (30 M).It is concluded that adenosine, at physiologically relevant concentrations, inhibits electrically evoked NA release from terminals in the central nervous system. Alkylxanthines increase evoked NA release from hippocampal terminals, wich probably not related to cyclic AMP but may partly involve inhibition of endogenous adenosine acting as a modulator of transmitter release in the hippocampal slice preparation.     

Quantitative freeze-fracture analysis of the frog neuromuscular junction synapse – II. Proximal–distal measurements

Neurotransmitter release from different parts of frog motor nerve terminals is often non-uniform. There is a decrease in release efficacy from the distal regions of frog motor nerve terminal branches. Since release is thought to occur near the double arrays of large intramembranous particles that constitute the pre-synaptic active zones (AZs), we have examined quantitatively the proximal–distal distribution of AZ structure, using a novel freeze-fracture technique that produces replicas of large fractions of terminals, including the region of nerve entry. This enables us to know the proximal–distal orientation of each branch. From 23 end-plates we have obtained fractures of 72 branches. For 27 of these branches we have obtained continuous fractures both greater than 25 μm in length and with sufficient information to determine their proximal–distal polarity. Only a few of these branches showed a marked distal decrease in AZ length/unit length of terminal, while several junctions had short regions (5–10 μm), either proximally or distally, that exhibited amounts of AZ that were substantially greater or smaller than the mean value for that terminal branch. The terminal area, post-synaptic gutter width and nerve terminal width all exhibit some distal decline concomitant with the distal tapering of nerve terminal branches. AZ length tends to have the least decline compared to the other parameters. Thus, the vast majority of frog motor nerve terminal branches do not display a significant proximal-distal gradient in the amount of AZ structure/μm terminal length. The present data do not provide an obvious ultrastructural correlate for the distal decline in transmitter release that some authors have observed.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

Endogenous Noradrenaline and Dopamine in Nerve Terminals of the Hippocampus: Differences in Levels and Release Kinetics

The presence and release of endogenous catecholamines in rat and guinea pig hippocampal nerve terminals was studied by fluorimetric HPLC analysis. In isolated nerve terminals (synaptosomes) the levels and breakdown of endogenous catecholamines were determined and the release process was characterized with respect to its kinetics and Ca2+ and ATP dependence. Endogenous noradrenaline and dopamine, but not adrenaline, were detected in isolated hippocampal nerve terminals. For dopamine both the levels and the amounts released were more than 100-fold lower than those for noradrenaline. In suspension, released endogenous catecholamines were rapidly broken down. This could effectively be blocked by monoamine oxidase inhibitors, Ca(2+)-free conditions, and glutathione. The release of both noradrenaline and dopamine was highly Ca2+ and ATP dependent. Marked differences were observed in the kinetics of release between the two catecholamines. Noradrenaline showed an initial burst of release within 10 s after K+ depolarization. The release of noradrenaline was terminated after approximately 3 min of K+ depolarization. In contrast, dopamine release was more gradual, without an initial burst and without clear termination of release within 5 min. It is concluded that both catecholamines are present in nerve terminals in the rat hippocampus and that their release from (isolated) nerve terminals is exocytotic. The characteristics of noradrenaline release show several similarities with those of other classical transmitters, whereas dopamine release characteristics resemble those of neuropeptide release in the hippocampus but not those of dopamine release in other brain areas. It is hypothesized that in the hippocampus dopamine is released from large, dense-cored vesicles, probably colocalized with neuropeptides.     

The mechanism of action of beta-bungarotoxin at the presynaptic plasma membrane.

The beta-bungarotoxin-induced depolarization of the synaptosomal plasma membrane monitored by the efflux of 86Rb+ is potentiated by raising the albumin in the incubation, is Ca2+-dependent and is due neither to inhibition of the (Na+ + K+)-dependent ATPase nor to activation of the voltage-dependent Na+ channel. Occupancy of the beta-bungarotoxin-binding site by dendrotoxin inhibits partially the action of beta-bungarotoxin. The efflux of 86Rb+ is parallelled by a release of lactate dehydrogenase from the synaptosome, and the two processes are maximal with 2 nM-toxin. Digitonin induces a release of 86Rb+ and lactate dehydrogenase closely similar to that seen with beta-bungarotoxin. It is concluded that the toxicity of beta-bungarotoxin for mammalian nerve terminals can be largely accounted for by specific site-directed phospholipase A2-induced permeabilization of the plasma membrane.     

An emerging view of presynaptic structure from electron microscopic studies

In response to calcium influx, some of the synaptic vesicles in presynaptic terminals fuse rapidly with the presynaptic membrane, allowing fast synaptic transmission. The regulated recycling of synaptic vesicles at the terminals is required for a sustained release of neurotransmitters. Localization of 'ready to be released' vesicles in close vicinities to voltage-gated calcium channels enables the rapid release of neurotransmitters. Thus, recycling vesicles must translocate from the sites of endocytosis to these release sites. However, the sub-cellular organization that supports this local vesicular traffic remains poorly understood. We will review the results of various electron microscopy studies, which have begun to unveil the structure of presynaptic terminals.     

Histochemical demonstration of transmitter release from noradrenaline,dopamine and 5-hydroxytryptamine nerve terminals in field stimulated rat brain slices

Summary The effect of electrical field stimulation on noradrenaline (NA), dopamine (DA) and 5-hydroxytryptamine (5-HT) nerve terminals in rat brain slicesin vitro was investigated. Slices prepared from the cerebral cortex or the neostriatum were incubated in physiologic buffer for 30 min and then superfused by buffer and stimulated by an electrical field (biphasic pulses, 10 Hz, 12 mA, 2 ms) for various time periods. The effect of the stimulation was studied at the cellular level with the histochemical fluorescence technique of Falck and Hillarp. The transmitter overflow into the superfusing buffer caused by the stimulation was studied with isotope technique. Cerebral Cortex NA Nerve Terminals. Stimulation caused release of NA from cortical NA nerve terminals. Already after 2 min stimulation a slight decrease of the fluorescence intensity of the nerve terminals could be found. Stimulation for 15 to 30 min greatly reduced the fluorescence intensity. In slices preincubated with³H-NA the stimulation-induced overflow of tritium during 2 min stimulation was about 15% (i.e. 15% of the tissue tritium content was overflowing into the superfusing buffer in response to stimulation for 2 min). During prolonged stimulation there was a considerable decline of the tritium efflux. Cerebral Cortex 5-HT Nerve Terminals. The 5-HT-analogue 6-hydroxytryptamine (6-HT) which is readily taken up into 5-HT nerve terminals was used to permit good visualization of these nerve terminals. Uptake of 6-HT into cortical NA nerve terminals was prevented by preincubation with 6-hydroxydopamine (6-OH-DA) or protriptyline. Stimulation for 15 to 30 min reduced the fluorescence intensity of the 5-HT nerve terminals. In slices preincubated with³H-5-HT the stimulation-induced overflow of tritium during 2 min stimulation was about 5%. The tritium efflux slowly decreased during continuous stimulation. Neostriatal DA Nerve Terminals. In slices frozen directly after preparation an intense diffuse fluorescence could be seen. After incubation in drug-free buffer at 37° C the fluorescence was localized in the varicosities of the DA nerve terminals. The central parts of the slices almost completely lacked specific fluorescence, while the outer zones were brightly fluorescent. No clear reduction of the fluorescence intensity of the DA nerve terminals in the outer zone could be observed after stimulation for 30 min. In slices preincubated with³H-DA the stimulation-induced overflow of tritium during 2 min stimulation was about 2%. The tritium efflux slowly decreased during continuous stimulation.It is suggested that the differences in release between the various nerve terminal systems foundin vitro reflect differences in transmitter release occurringin vivo. The comparably high release of NA per impulse from the cortical NA nerve terminals implicate that the discharge rate of these neuronsin vivo is very low.This investigation has been supported by grants from the Swedish Medical Research Council (B72-14X-2330-05A) and Magnus Bergwall's Foundation.The author is greatly indebted to Mrs. Annika Hamberger for her skillful technical assistance. For generous supplies of drugs thanks are due to Hässle, Göteborg, Sweden, through Dr. H. Corrodi (6-HT, 6-OH-DA and H44/68).     

Release of Monoamines from Striatum of Rat and Mouse Evoked by Local Application of Potassium: Evaluation of a New In Vivo Electrochemical Technique

Local application of K+ via micropressure-ejection, coupled with in vivo electrochemical detection, was used to study stimulated release from monoaminergic nerve terminals in the striatum of anesthetized rats and mice. K+-evoked releases were reversible, reproducible, and dose-dependent. In contrast, releases of electroactive species could not be evoked by local ejection of Na+. The magnitudes and time courses of K+-evoked releases recorded from the caudate nucleus of mice were greater than those seen in rats. Local application of nomifensine, a putative catecholamine reuptake blocker, augmented the magnitudes and time courses of K+-evoked releases. Releases were also recorded from brain regions adjacent the striatum; these signals were always smaller than those seen in the caudate nucleus and had amplitudes that showed good correspondence to the relative degree of dopaminergic input to these areas. These data, taken together with other information in the literature, suggest that this new technique is well suited for in situ studies of monoamine release and reuptake in intact animals.     

Presynaptic actions of botulinal neurotoxins at vertebrate neuromuscular junctions

1. In the present paper we review some presynaptic aspects of the mode of action of botulinal toxins (BoTxs) at vertebrate neuromuscular junctions with emphasis on studies carried out in our laboratories using electrophysiological and morphological techniques. 2. Spontaneous quantal transmitter release recorded as miniature end-plate potentials is drastically affected by BoTxs. The low probability of release at poisoned terminals can be enhanced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), Cd2+ and La3+. However, CCCP and La3+ which drastically deplete clear synaptic vesicles from unpoisoned terminals failed to markedly affect the density of synaptic vesicles at poisoned terminals. It is concluded that poisoned terminals have a reduced sensitivity to the release-promoting action of Ca2+, Cd2+ and La3+. 3. When comparing the effect of the various BoTxs on nerve-impulse evoked transmitter release it appears that increasing phasic Ca2+ entry into the terminals enhances evoked synchronized quantal release only from terminals poisoned with serotypes A and E. In contrast, enhanced Ca2+ entry into terminals poisoned with serotypes B, D and F induced a period of high frequency asynchronous release suggesting that these BoTxs may affect a presynaptic step beyond the influx of Ca2+, that may be involved in the synchronization of transmitter quanta. These data suggest that the actions of BoTxs involve several steps of the acetylcholine release process. 4. The analysis of presynaptic currents which depend on both Ca2+ entry and intraterminal background Ca2+ levels strongly suggests that neither Ca2+ entry nor intraterminal Ca2+ levels are altered by BoTxs. Furthermore, poisoned terminals are no more efficient than unpoisoned ones in dealing with Ca2+ overloads. 5. Finally, the morphological examination of junctions paralysed by BoTx-A indicates that the toxin triggers a particularly important overgrowth of the nerve terminals and suggests that the in vivo functional recovery may occur from an extension of the original nerve terminal arborization and the concomitant remodelling of postsynaptic structures.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Differential modulation of N‐type calcium channels by µ‐opioid receptors in oxytocinergic versus vasopressinergic neurohypophysial terminals

Opioids modulate the electrical activity of magnocellular neurons (MCN) and inhibit neuropeptide release at their terminals in the neurohypophysis. We have previously shown that µ‐opioid receptor (MOR) activation induces a stronger inhibition of oxytocin (OT) than vasopressin (AVP) release from isolated MCN terminals. This higher sensitivity of OT release is due, at least in part, to the selective targeting of R‐type calcium channels. We now describe the underlying basis for AVP's weaker inhibition by MOR activation and provide a more complete explanation of the complicated effects on neuropeptide release. We found that N‐type calcium channels in AVP terminals are differentially modulated by MOR; enhanced at lower concentrations but increasingly inhibited at higher concentrations of agonists. On the other hand, N‐type calcium channels in OT terminals were always inhibited. The response pattern in co‐labeled terminals was analogous to that observed in AVP‐containing terminals. Changes in intracellular calcium concentration and neuropeptide release corroborated these results as they showed a similar pattern of enhancement and inhibition in AVP terminals contrasting with solely inhibitory responses in OT terminals to MOR agonists. We established that fast translocation of Ca²⁺ channels to the plasma membrane was not mediating current increments and thus, changes in channel kinetic properties are most likely involved. Finally, we reveal a distinct Ca‐channel β‐subunit expression between each type of nerve endings that could explain some of the differences in responses to MOR activation. These results help advance our understanding of the complex modulatory mechanisms utilized by MORs in regulating presynaptic neuropeptide release. J. Cell. Physiol. 225: 276–288, 2010. © 2010 Wiley‐Liss, Inc.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

The cytoskeletal lattice of the neurohypophysial cells

The cytoskeleton of rat neurohypophysial cells as seen in resinless sections is an irregular three-dimensional lattice of short strands of cytoplasmic matrix (the microtrabeculae) that interconnect parallel arrays of neurotubules, neurofilaments, abundant neurosecretory granules, and other membrane-bound organelles including the plasma membrane. This morphological finding suggests that the cytoplasmic ground substance constitutes a cytoskeletal continuum that may be the ultrastructural expression of a motile apparatus responsible for neurosecretory granule movement and hormone release in the neurohypophysis.     

Subtype-specific expression of group III metabotropic glutamate receptors and Ca2+ channels in single nerve terminals

The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca(2+) channel activity. By imaging Ca(2+) in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca(2+) channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca(2+) channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for l(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca(2+) response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca(2+) channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca(2+) channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca(2+) channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.