Vesicle-associated proteins and P2X receptor clusters at single sympathetic varicosities in mouse vas deferens.

1. Antibodies against vesicle-associated proteins of the SNARE complex (syntaxin (AbS), SNAP 25 (AbS25), synaptobrevin (VAMP; AbV) and the alpha1B subunit of calcium channels (Abalpha(1B)) were located with respect to sympathetic varicosities (labelled with the ubiquitous vesicle proteoglycan antibody AbSV2) and to clusters of P2X receptor subunits (labelled with antibodies AbP2X(1) to AbP2X(6)). In addition, these receptor clusters were located with respect to Schwann cells labelled with antibodies to S100 (AbS100).2. The spatial relation between proteins of the SNARE complex and calcium channels was determined. AbS25 patches ranged from 250-500 nm in size and were often colocalised with smaller AbS patches (250-350 nm). Abalpha(1B) patches (300-700 nm diameter) were always coincidental with AbS patches. AbV patches (400-1000 nm in diameter) also coincided with AbS patches.3. The spatial relation between different P2X subunit clusters and varicosities labelled with AbSV2 was ascertained. Large (500-700 nm diameter) AbP2X(1) receptor clusters were found colocalised with many (91%) AbSV2 labelled varicosities, although small diameter (250-350 nm) AbP2X(1) clusters occurred at random over the muscle. Small AbP2X(2) clusters were found uniquely in the vicinity of AbSV2 labelled varicosities, but were not entirely coincidental with these. Small AbP2X(3) receptor clusters were found in close association with AbSV2 labelled nerves. Small diameter AbP2X(4) clusters (250-350 nm) were found throughout the muscle with some of these coincidental with AbSV2 labelling. Small diameter AbP2X(5) (250-350 nm) cluster labelling was found in juxtaposition to strings of AbSV2 labelled varicosities but were not coincidental with these. Small (250-350 nm) diameter AbP2X(6) clusters were also found in close juxtaposition to AbSV2 labelled nerves.4. The spatial relation between different P2X subtype clusters and Schwann cells labelled with AbS100 was examined. Both AbP2X(1) and AbP2X(3) receptor clusters were found in close apposition with AbS100, with clusters of the former sometimes coincidental with patches of the latter. On the other hand AbP2X(2) was found in association with AbS100 at low levels while AbP2X(4) labelling was generally not coincidental with AbS100. AbP2X(5) and AbP2X(6) labelling was often colocalised with AbS100 labelling.5. The spatial relation between proteins of the SNARE complex and P2X(1) receptors was determined. Large AbP2X(1) clusters were often found apposed by AbS, AbV and Abalpha(1B) labelled patches.6. Destruction of the sympathetic varicosities with 6-hydroxydopamine led to the virtual disappearance of AbP2X(2) labelling, but to a large increase in the number of small AbP2X(1) receptor clusters and a reduction in the number of large AbP2X(1) clusters. AbS100 Schwann cell labelling was largely unaffected.7. These observations are interpreted as showing that most terminal sympathetic varicosities possess active zones about 250-700 nm diameter, delineated by syntaxin, SNAP 25 and N-type calcium channels and that synaptic vesicles are concentrated at these sites as indicated by the localisation of VAMP. Most of these terminal varicosities possess active zones that are precisely apposed to large clusters of P2X(1) receptors. However small clusters of P2X(2) to P2X(6) receptors can be found that are near the strings of varicosities but not usually coincidental with them except P2X(3). The functional significance of this arrangement of vesicle-associated proteins and P2X receptors for the generation of synaptic potentials at the autonomic neuromuscular junction is discussed.

Varicosities of single sympathetic nerve terminals possess syntaxin zones and different synaptotagmin N-terminus labelling following stimulation.

A study has been made of the probability of exocytosis of synaptic vesicles at different varicosities in single sympathetic terminal axons in the mouse was deferens. An antibody (SV2Ab) against SV2. a proteoglycan in synaptic vesicles, labelled an area of individual sympathetic varicosities that was slightly less than that occupied by dextran-rhodamine, previously orthogradely transported into the varicosities. In contrast plasma membrane bound protein syntaxin, found at active zones of motor nerve terminals, occupied an area of the varicosity that was approximately one-third that of SV2. This suggests that sympathetic varicosities possess specialized zones for exocytosis on their plasma membranes. Antibodies against the N-terminal sequence of synaptotagmin 1 (SNAb), a sequence exposed within synaptic vesicles, were used to determine the probability of exocytosis at different varicosities of single terminal branches. The area of SNAb labelling was not significantly different from that of the SV2 labelling, which implies vesicles that have undergone exocytosis may eventually return to the main pool of vesicles. Varicosities belonging to the same terminal axon, and identified with SV2Ab, showed different extents of labelling with SNAb when secretion was evoked with high potassium concentrations (80 mM) for 30 min in the presence of SNAb. There was up to an order of magnitude difference in the average intensity of SNAb labelling between different varicosities of the same terminal axon whereas there was little difference in the average intensity of SV2Ab labelling. These observations suggest that there is considerable variability in the probability of exocytosis at the specialized zones in different varicosities.

Spatial relationships between sympathetic varicosities and smooth muscle cells in the longitudinal layer of the mouse vas deferens.

The spatial relationships between nerve varicosities and smooth muscle cells in the longitudinal muscle layer of the mouse vas deferens have been determined from serial section reconstructions of individual varicosities at the ultrastructural level. Bundles of up to five axons, together with single axons, occurred frequently at the surface of the muscle as well as at about 3-6 muscle cell diameters into the muscle. Varicosities within axon bundles at the muscle surface each became partially divested of Schwann cell processes. The smallest distance separating varicosity membrane from muscle cell membrane (apposition distance) was 100 nm (mean 170 nm) for varicosities contained in bundles. Varicosities from six single axons on the muscle surface were reconstructed and 11 of the 12 possessed a mean apposition distance of 48 nm. Varicosities in axon bundles at about 12 microns deep into the muscle came into an apposition distance of 50-90 nm (mean = 67 nm). All varicosities of single axons at this depth came into about 50 nm apposition (mean = 53 nm). These results indicate that the varicosities lie at varying distances from the muscle cells in the longitudinal muscle layer of the vas deferens.

Function of the Y optic nerve fibres in the cat: do they contribute to acuity and ability to discriminate fast motion?

1. A controlled pressure block has been applied to the optic nerve of the cat, sufficient to bring about degeneration of the axons of the large (Y) nerve fibres caudal to the block site. This degeneration has been monitored by means of implanted electrodes in optic nerve and tract which have shown a loss of the short-latency (t1) response 4-6 days after the block, and also by histological examination of the optic nerve. 2. Cats with one optic nerve blocked in this way have been used in behavioural experiments, one or other eye being covered during the tests. Tested via the blocked nerve, all cats with loss of only Y fibres could perform certain tests: the visual placing reaction, the blink reflex, the pupillary (light) reflex and simple manoeuvres such as walking a plank and jumping from table to floor. 3. When acuity was tested by means of the Mitchell jumping apparatus, cats with loss of only Y fibres showed the same acuity using either eye. This was true also of one cat in which many X fibres had also degenerated, as evidenced by a 55% loss of the medium-latency (t2) response, but in another cat with 90% loss of the t2 response acuity was reduced to about half-normal. 4. Ability to discriminate fast motion was tested by a modification of the Mitchell apparatus. All cats were able to discriminate the motion of an 11.5 deg spot up to a velocity of 6260 deg/s, whether using their normal eye or their affected eye. However, the loss of the Y fibres reduced the ability to discriminate fast motion, so that for any given level of contrast the velocity which could be discriminated was about two-thirds of the velocity discriminated using the normal eye. The ability of the cat to discriminate fast motion seems to be similar to that of the human. 5. These results suggest that there is no sharp restriction of function between the Y and X systems but instead considerable overlap. However, each system possesses specialized features giving it superiority in certain conditions.

Selective degeneration of optic nerve fibres in the cat produced by a pressure block.

Using a technique described previously, we have applied pressure to the optic nerve of a cat sufficient to cause conduction block of the t1 response (the response of the Y optic nerve fibres). A greater pressure, usually sufficient to cause a transient block of the t2 response (the response of the X fibres), leads to degeneration of the Y axons caudal to the block. This is demonstrated by the disappearance of the t1 response in this region after 4-5 days and by the presence in electron micrographs of degenerating large (Y) fibres. Some small fibres also show degeneration, but the medium (X) fibres are largely spared. The time course of loss of response in the Y fibres is similar whether the loss is due to a pressure block or to enucleation, suggesting that the pressure block as used by us causes a disruption of the axon. If the pressure is great enough to block part of the t2 response (X fibres) there is also a similarity in time course of loss of response to that following enucleation. Both for the enucleated and the pressure-blocked cat the t2 response fails about 1 day before the t1 response. This is in apparent disagreement with the morphological findings in the literature, confirmed here, indicating an earlier degeneration of the larger fibres. The post-synaptic response in the lateral geniculate nucleus to the t1 input (the r1 response) also fails about 1 day before the t1 response. In the visual cortex the loss of the r1 response reveals more clearly than is normally possible an r2 response, the response of the X optic radiation fibres. The response in the optic nerve or tract to a bright flash of light is dominated by the response of the Y fibres. When these are blocked the response is greatly reduced.

Interrelations of the rat's thalamic reticular and dorsal lateral geniculate nuclei.

Electrophysiological and neuroanatomical techniques have been used to study the properties of cells in the reticular nucleus of the thalamus (RNT) responsive to photic stimuli. In the rat these cells are located in a discrete region of the nucleus lying immediately rostral to the dorsal lateral geniculate nucleus (LGNd), where the visual field is represented in a retinotopic fashion. After injections of horseradish peroxidase (HRP) into this area, neurones labelled with reaction product were found in the LGNd and not in other thalamic relay nuclei. After HRP injections into the LGNd, labelled RNT cells were found only within the region which contains neurones responsive to photic stimuli. These observations suggest that there is a precise reciprocal relation between the two areas. Studies and comparisons of the responses of relay cells (P cells) in LGNd and cells in RNT to electrical shocks lead us to conclude that RNT cells receive their excitation mainly via those relay cells in LGNd which are themselves excited by fast-conducting retinal ganglion cell axons. Such cells in LGNd have phasic responses and concentric receptive fields while RNT cells have phasic responses and on/off fields and a comparison of the receptive field sizes of P cells and RNT cells suggests that only a small number of LGNd relay cells converge on to each RNT cells. Further, although a particular functional class of relay cells in LGNd (Y-type) is shown to provide the major input to visually responsive RNT cells, both Y type and W type relay cells are subject to their inhibitory control. These results furnish evidence that cells in the RNT have an important role in modulating the flow of visual information from the LGNd to cortex.

Cortical projections to visual centres in the rat: an HRP study.

We have investigated the relationships of the visual cortex to other visual centres in the rat: namely the lateral geniculate nucleus, the visually responsive part of the thalamic reticular nucleus and the superior colliculus. We injected horseradish peroxidase iontophoretically so as to restrict the injectate to each of the regions, and reacted sections using 3 different procedures. Areas 17, 18 and 18a project to both dorsal and ventral lateral geniculate nucleus as well as to the visually responsive part of the thalamic reticular nucleus and superior colliculus. Pyramidal cells in lamina VI project to the dorsal lateral geniculate nucleus and to the thalamic reticular nucleus, whereas cells of origin of the projection to the superior colliculus lie in lamina V; cells in lamina V also project to ventral lateral geniculate nucleus. The implications of these findings are discussed, particularly in terms of the functional relationships between the visual cortex, lateral geniculate nucleus and visual thalamic reticular nucleus.

Retrograde intraaxonal transport of horseradish peroxidase by neurons in octopus.

While retrograde axonal transport is the basis of a widely used neuroanatomical method, it has been rigorously demonstrated in vivo only in a few vertebrate species and not yet in an invertebrate. Evidence is presented that motor neurons of the octopus stellate ganglion are capable of retrograde intraaxonal transport of horeseradish peroxidase. This demonstration shows that retrograde transport occurs in widely divergent groups of animals, and may be a general property of neurons.

Visual receptive-field properties of cells in area 18 of cat's cerebral cortex before and after acute lesions in area 17.

1. Receptive-field properties of single neurons in cat's cortical area 18 were studied before and after partial bilateral lesions of area 17. 2. The majority of cells recorded from animals with intact visual cortex exhibited orientation selectivity, directional selectivity, and could be independently activated through either eye. All cells responded well to moving targets and nearly all of them exhibited broadly tuned preferences with respect to speed of the target. Over 45% of cells responded optimally or exclusively at very fast (above 50 degrees/s) speeds. 3. The majority of neurons recorded from animals with intact visual cortex responded weakly but clearly to appropriately oriented localized stationary stimuli flashed on and off. About one-third of the cells responded with mixed on-off discharges from all over their receptive field. In the receptive fields of 10% of cells, separate on- and off-discharge regions could be revealed. In the receptive fields of the remaining cells, only on- or only off-discharge regions could be revealed. 4. The majority of neurons recorded after ablation of area 17 were orientation selective; 50% of the cells were also direction selective. All neurons responded well to moving targets; about 65% of them responded optimally or exclusively at very fast target speeds. 5. Destruction of the dorsolateral part of contralaterial area 17 and most of contralateral area 18 caused significant reduction in proportion of cells in area 18 which could be activated through either eye. 6. The majority of neurons recorded after ablation responded to appropriately oriented localized stationary stimuli flashed on and off. Cells with mixed on-off discharge regions all over the receptive field with separate on- and off-discharge regions and with only on- or only off-discharge regions were found. 7. It is concluded that the processing of afferent visual information in area 18 is, to a great extent, independent of the information carried to this area by associational fibers from cells of area 17.