John T. Schmidt

Activity sharpens the map during the regeneration of the retinotectal projection in goldfish

In the regenerating retinotectal projection of goldfish, we have used intraocular injections of tetrodotoxin (TTX) to determine whether activity plays a role in organizing or refining the retinotopic map. Repeated injections produced a continuous 27-day block without producing extraocular effects or causing deleterious effects in the retinal ganglion cells. The retinotectal maps regenerated in the TTX fish were normally organized but the multiunit receptive fields were grossly enlarged. In control regenerates, 1-3 units (arbors of retinal ganglion cell axons) were simultaneously recorded at each penetration and their combined receptive field averaged 11-12 degrees, nearly the same as for single units. In TTX fish each penetration yielded at least 5-10 units whose receptive fields were clustered over a wider area averaging 27 degrees across. Individual ganglion cell receptive fields were assessed both by tectal and by intraretinal recording and were not enlarged. Many fish were recorded up to 4 months after the release from TTX block, but no further refinement of the maps occurred. If the nerve was recrushed and regenerated a second time without TTX, a normal map was formed, ruling out any permanent changes in the retinal ganglion cells or in the tectum. Blocks during various portions of the regeneration process showed that lack of activity during the process of axonal elongation (first 2 weeks) does not cause enlargement of the multiunit receptive fields, but lack of activity during the period of synapse formation and maturation (14-34 days) does. The results are discussed in terms of an activity-dependent stabilization of synapses. Neighboring retinal ganglion cells are known to fire in a statistically correlated fashion and this could help in their elimination of incorrect branches following an early period of diffuse connections.

Videofluoroscopic evidence of aspiration predicts pneumonia and death but not dehydration following stroke

In order to assess the risk of pneumonia, dehydration, and death associated with videofluoroscopic evidence of aspiration following stroke, the clinical records of 26 patients with aspiration and 33 randomly selected, case-matched, dysphagic controls without videofluoroscopic evidence of aspiration were reviewed. The videofluoroscopic modified barium swallow technique included 5 ml-thin and thick liquid barium, 5 ml barium pudding, and 1/4 cookie coated with barium, plus additional 20 and 30 ml of thin liquid barium. Patients were assessed a mean of 2±1 SD months poststroke and were followed for a mean of 16±8 SD months poststroke. The odds ratio for developing pneumonia was 7.6 times greater for those who aspirated any amount of barium irrespective of its consistency (p=0.05). The odds ratio for developing pneumonia was 5.6 times greater for those who aspirated thickened liquids or more solid consistencies compared with those who did not aspirate, or who aspirated thin liquids only (p=0.06). Dehydration was unrelated to the presence or absence of aspiration. The odds ratio for death was 9.2 times greater for those aspirating thickened liquids or more solid consistencies compared with those who did not aspirate or who aspirated thin liquids only (p=0.01). Aspiration documented by modified videofluoroscopic barium swallow technique is associated with a significant increase in risk of pneumonia and death but not dehydration following stroke.

Stroboscopic illumination and dark rearing block the sharpening of the regenerated retinotectal map in goldfish

Blocking activity with intraocular tetrodotoxin prevents the sharpening of the retinotectal map formed during regeneration of the optic nerve. If (under normal conditions) the initially diffuse map sharpens because of correlated activity in neighboring but not distant ganglion cells, then sharpening should also be prevented merely by disrupting the spatiotemporal correlation in the pattern of activity. To test this idea, fish were exposed during regeneration to stroboscopic illumination in a featureless environment, or were maintained in complete darkness. The regenerating cells remained visually responsive after axotomy, and the xenon strobe effectively drove each ganglion cell at a constant latency. The maps formed in the strobe-reared fish were normally oriented, but the multiunit receptive fields were greatly enlarged, averaging 32 degrees. In control regenerates, multiunit receptive fields averaged only 11-12 degrees, nearly the same as for single units. Dark rearing, which allows only spontaneous activity, also resulted in enlarged multiunit receptive fields, averaging more than 28 degrees. Both effects parallel those reported previously with tetrodotoxin block. The mature projection did not become diffuse as a result of the strobe rearing, and the sensitive period corresponded to the early stage of synaptogenesis (20-34 days). Periods of normal visual exposure after 35 days produced very little sharpening of the diffuse maps produced during either strobe or dark rearing. The results are attributed to an activity-dependent stabilization of developing synapses. The correlated firing of neighboring ganglion cells could allow postsynaptic summation of their responses, and the retention of those more effective, retinotopically placed synapses might then occur via a Hebbian mechanism.

The re-establishment of synaptic transmission by regenerating optic axons in goldfish: Time course and effects of blocking activity by intraocular injection of tetrodotoxin

Intraocular injections of tetrodotoxin were used to block activity for 27 days in normal fish and for the first 27 or 31 days of regeneration in fish with one optic nerve crushed. Synaptic activity was then assessed by a current source-density analysis of field potentials evoked by optic nerve shock at different times following the TTX treatment. In normal fish, the lack of activity for 4 weeks had no significant effect on the maintenance of synaptic strength. Likewise, in fish with nerve crush, lack of activity did not prevent the regenerating optic fibers from forming synapses that were nearly as effective as those formed in controls injected with the citrate buffer vehicle. The earliest synapses were formed at the rostromedial corner of the tectum (where the tract enters) at 20 days after nerve crush, when fibers had not yet reached the caudal areas. By 28 days synaptic potentials could be recorded everywhere on the surface of the tectum in both controls and TTX injected fish. However, the latency of the responses with TTX were longer, suggesting a smaller caliber of fiber, which is consistent with an earlier finding of decreased axonal transport in TTX fish. Maturation of the regenerating fibers proceeded slowly in both TTX and control fish. After more than 5 months, the projections were nearly normal but still not completely normal.

Activity and the formation of ocular dominance patches in dually innervated tectum of goldfish

This study tested (1) whether blocking impulse activity in both eyes of fish with one tectum prevents the formation of ocular dominance patches, (2) whether areas receiving a high density of innervation from one eye receive a low density from the other, and (3) whether there is an electrophysiological correlate to the anatomical patches. One tectum was removed in goldfish so that the optic nerve fibers from both eyes would compete for synaptic space in the remaining tectum. The terminal arbors from the two projections initially overlapped but by 50 to 60 days segregated into ocular dominance patches, demonstrated by labeling both projections, the normal one with horseradish peroxidase and the regenerating one with tritiated proline radioautography. Alternate sections were processed for radioautography and histochemistry. All projections were drawn by “blind” observers using a camera lucida and were fully reconstructed. Both the level of patchiness within each projection and the correspondence of patches and holes between the two projections were quantified from these reconstructions. Binocular tetrodotoxin (TTX) injections from 18 to 75 days after surgery significantly reduced patchiness, as compared to controls injected with citrate-Ringers solution. When the binocular block was continued until 95 days, segregation was still significantly reduced relative to controls. These results support a hypothesis for an activity-dependent mechanism of segregation of ocular dominance patches. In controls but not TTX-blocked fish, there was a significant tendency for high density areas in one projection to receive a lower density projection from the other eye, and vice versa. However, the two projections were not entirely complementary. Survival of control fish for an additional 5 months resulted in more sharply defined patches but no increase in complementarity. Recordings of field potentials evoked by shocking either optic nerve demonstrated an electrophysiological correlate to the anatomical patches in single tectal fish. Large field potentials from one eye were generally associated with small potentials from the other eye, and vice versa. When the recording sites were marked with electrolytic lesions, there was a direct and significant correlation between the magnitude of the field potentials and the density of the anatomical ocular dominance patches.

Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.

Summary1.During regeneration of the optic nerve in goldfish, the ingrowing retinal fibers successfully seek out their correct places in the overall retinotopic projection on the tectum. Chemospecific cell-surface interactions appear to be sufficient to organize only a crude retinotopic map on the tectum during regeneration.2.Precise retinotopic ordering appears to be achieved via an activity-dependent stabilization of appropriate synapses and is based upon the correlated activity of neighboring ganglion cells of the same receptive-field type in the retina. Four treatments have been found to block the sharpening process: (a) blocking the activity of the ganglion cells with intraocular tetrodotoxin (TTX), (b) rearing in total darkness, (c) correlating the activation of all ganglion cells via stroboscopic illumination and (d) blocking retinotectal synaptic transmission withα-bungarotoxin (αBTX).3.These experiments support a role for correlated visually driven activity in sharpening the diffuse projection and suggest that this correlated activity interacts within the postsynaptic cells, probably through the summation of excitatory postsynaptic potentials (EPSPs).4.Other experiments support the concept that effective synapses are stabilized: a local postsynaptic block of transmission causes a local disruption in the retinotectal map. The changes that occur during this disruption suggest that each arbor can move to maximize its synaptic efficacy.5.In development, initial retinotectal projections are often diffuse and may undergo a similar activity-dependent sharpening.6.Indirect retinotectal maps, as well as auditory maps, appear to be brought into register with the direct retinotopic projections by promoting the convergence of contacts with correlated activity.7.A similar mechanism may drive both the formation of ocular dominance patches in fish tectum and kitten visual cortex and the segregation of different receptive-field types in the lateral geniculate nucleus.8.Activity-dependent synaptic stabilization may therefore be a general mechanism whereby the diffuse projections of early development are brought to the precise, mature level of organization.

MK801 increases retinotectal arbor size in developing zebrafish without affecting kinetics of branch elimination and addition

Visual activity refines the retinotopic map formed on tectum during regeneration and development in goldfish through an N-methyl-D-aspartate (NMDA) receptor-mediated mechanism. Retinal arbors are enlarged in fish with unrefined maps. Here, we examined the effect of NMDA receptor blockers on the development of retinotectal arbors in zebrafish. Since visual behaviors begin 68-79 h postfertilization, we blocked NMDA receptors by immersion of larvae in MK801, AP5, or CPP starting at either 48 or 72 h. We then labeled axons with DiI at 72 or 96 h and examined them 5-9 h later. Arbors at 101-105 h (31 cases) were larger than at 77-79 h (11 cases): The average number of branches increased from 4.0 to 7.6 and the area (convex polygon method) increased by 42%. Blocking NMDA receptors with MK801 from 72 to 101-105 h significantly enlarged arbor size, but the number of branches remained roughly the same. The length and area of the arbors were both significantly increased (21% and 36%), whereas the width increased by a smaller amount (6%). This increase was reflected in longer distances between branches within the arbor (interbranch segments, +13%) as well as in the summed length of all branches (+28%). This selective effect on the extent but not number of branches is in agreement with our previous report of strobe effects in both developing and regenerating projections in goldfish, and supports the role of NMDA receptors in the first 24 h of synaptic transmission. We also used DiO to label arbors in time-lapse images taken at hourly intervals from 77 to 112 h. These sequences confirmed that individual arbors grew during this time, but showed that rates of branch addition and deletion and branch lifetimes were unaltered by the MK801 treatment. This is consistent with a simple model of random insertion of new branches and selective activity-driven elimination of those at the periphery to keep the normal arbor focused. Blocking NMDA receptors is postulated to randomize the elimination allowing the periphery to expand, thus accounting for the enlarged areas, without change in branch numbers or branch dynamics.

Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish

The regenerating optic nerve of goldfish first reestablishes a rough retinotopic map on the tectum, then goes through an activity dependent refinement that appears to involve the elimination of inappropriate branches from early regenerated arbors. Retinotopically appropriate branches and synapses may be stabilized because the normally correlated firing of neighboring ganglion cells could cause summation of their postsynaptic responses, making them more effective. Thus, refinement of the map may be similar in several ways to associative learning. In this study, we therefore tested whether ependymin, a major protein component of the extracellular fluid that has been implicated in synaptic changes thought to be associated with learning a simple task in goldfish, may also be involved in refinement of the retinotopic map. Goldfish that had undergone unilateral optic nerve crush received intraventricular infusion of antiependymin IgG or of control IgGs beginning at 21 days postcrush. Tectal recordings from these fish at 39-56 days postcrush showed that the projection had failed to sharpen, much as in the fish with activity blocked or synchronized; the average size of the multiunit receptive fields was 31 degrees vs 11 degrees normally. The field potentials elicited from these tecta by optic nerve shock were not significantly smaller than in controls, suggesting normal levels of synaptogenesis. Control projections, identically treated but infused with either unrelated IgG or Ringers alone regenerated normally, giving multiunit receptive fields of 12 degrees. Intact (non-regenerating) projections of the experimental fish were not rendered abnormal by the IgG treatment. Histology showed the retinas and tecta of the infused fish to be normal in appearance. The results show a specific block of sharpening by antiependymin IgG. The ependymal glia of the tectum stain positively for ependymin in normal fish, particularly the cell bodies in the ependymal layer. The tectum, particularly the ependymal layer, stains more intensely during regeneration, which appears to trigger increased synthesis of ependymins in the ependymal glia. This increase and the block of sharpening by specific antibodies to ependymin suggest a possible role for ependymin in activity dependent synaptic stabilization, possibly through its polymerization when calcium is focally depleted at active synapses.

Eye-specific segregation of optic afferents in mammals, fish, and frogs: The role of activity

Summary1.Eye-specific patches or stripes normally develop in the visual cortex and superior colliculus of many (but not all) mammals and are also formed, after surgically produced binocular innervation, in the optic tectum of fish and frogs. The segregation of ocular dominance patches or columns has been studied using a variety of anatomical pathway-tracing techniques, by electrophysiological recording of postsynaptic units or field potentials, and by the 2-deoxyglucose method following visual stimulation of only one eye.2.In the tectum of both fish and frogs and in the cortex and colliculus of mammals, eye-specific patches develop from initially diffuse, overlapping projections.3.Of the various mechanisms that might cause such segregation, the evidence favors an activity-dependent process that stabilizes synapses from the same eye because of their correlated activity. First, several environmental manipulations affect the segregation of afferents in visual cortex: strabismus and alternate monocular exposure apparently enhance segregation, whereas dark rearing slows the segregation process, and monocular deprivation causes the experienced eye to form larger patches at the expense of those of the deprived eye. Second, blocking activity in both eyes is effective in preventing the segregation both in the tectum of fish and frog and in the visual cortex of cat. With the eyes blocked, alternate stimulation of the optic nerves permits the segregation of ocular dominance, at least onto single cells in the cat visual cortex.4.These findings are discussed in terms of an activity-dependent stabilization of those synapses having correlated activity (those from neighboring ganglion cells within one eye) but not of those lacking correlated activity (those from left and right eyes). We suggest that the eye-specific patches represent a compromise between total segregation of the projections from the two eyes and the formation of a single continuous retinotopic map across the surface of the cortex or tectum.

The long latency component of retinotectal transmission: Enhancement by stimulation of nucleus isthmi or tectobulbar tract and block by nicotinic cholinergic antagonists

The optic tectum of teleosts contains high concentrations of nicotinic and muscarinic acetylcholine receptors and receives putative cholinergic innervation from both nucleus isthmi in the tegmentum and a population of intrinsic tectal cells. Using in vitro preparations of goldfish brain, we have examined the effects of cholinergic antagonists and stimulation of nucleus isthmi on the tectal response to optic nerve stimulation. Our results show that: (1) a long latency component of the retinotectal field potential is polysynaptic in origin and occurs in isolated tectum; (2) this component can spread across the tectum from a beam of stimulated fibers and can appear in areas where the monosynaptic response is small or absent; (3) both monosynaptic and long latency components of the field potential are enhanced by prior stimulation of nucleus isthmi or the tectobulbar tract (15-300 ms); (4) both the long latency component of the field potential and the effects of stimulation of nucleus isthmic or tectobulbar tract are blocked by low concentrations of nicotinic antagonists; and (5) in deeper tectum a second polysynaptic response uncovered by pharmacological block of inhibition is not blocked by nicotinic antagonists. These results indicate that the cholinergic neurons intrinsic to tectum have a role in the spread of retinotectal excitation by nicotinic actions, and that stimulation of nucleus isthmi or tectobulbar tract facilitates activity in this system. There is in addition a separate recurrent excitatory circuit in tectum.