Callosal patches in primary visual cortex of Long Evans rats, normally associated with ocular dominance columns, emerge by postnatal day 10 (P10), but they do not form in rats monocularly enucleated a few days before P10. We investigated whether we could replicate the results of monocular enucleation by using tetrodotoxin (TTX) to block neural activity in one eye, or in primary visual cortex. Animals received daily intravitreal (P6–P9) or intracortical (P7–P9) injections of TTX, and our physiological evaluation of the efficacy of these injections indicated that the blockade induced by a single injection lasted at least 24 h. Four weeks later, the patterns of callosal connections in one hemisphere were revealed after multiple injections of horseradish peroxidase in the other hemisphere. We found that in rats receiving either intravitreal or cortical injections of TTX, the patterns of callosal patches analyzed in tangential sections from the flattened cortex were not significantly different from the pattern in normal rats. Our findings, therefore, suggest that the effects of monocular enucleation on the distribution of callosal connections are not due to the resulting imbalance of afferent ganglion cell activity, and that factors other than neural activity are likely involved.

Direction-selective responses were recorded from neurons in the dorsal terminal nucleus (DTN) of the cat accessory optic system before and after intravitreal injections of tetrodotoxin (TTX) into the contralateral eye. After approximately 100 min, direction-selective responses driven through stimulation of the contralateral, injected eye were reduced on average by 90%, while direction-selective responses driven through stimulation of the ipsilateral, uninjected eye were not significantly reduced. By 200 min postinjection, ipsilateral direction-selective responses were either equal to or sometimes greater than control values. In the final stages of these experiments (i.e. between 390–830 min after contralateral eye injections), ipsilateral eye responses were on average 30% higher than control. The effects of retinal blockade of the contralateral eye by TTX show that input from the ipsilateral eye alone is sufficient to mediate direction-selective responses in DTN cells. These results and those observed following bicuculline eye injections reported previously (Grasse et al. 1990) demonstrate that direction-selective responses in the DTN driven through stimulation of the contralateral and ipsilateral eyes arise from independent neural mechanisms located in the retina and visual cortex, respectively. Moreover, these findings also suggest that the contralateral eye exerts an inhibitory influence over ipsilateral eye responses which is diminished by TTX injections into the contralateral eye.

The laminar structure and cellular distribution of cytochrome-oxidase (CO) reactivity in supragranular puffs of striate cortex was examined in adult macaque monkeys surviving various periods of monocular enucleation, lid suture, and retinal impulse blockage with tetrodotoxin (TTX). Enucleation and TTX produced a rapid and severe loss in the size of the CO reactive region in puffs dominated by the removed or treated eye compared to slower and less marked reductions obtained in deprived puffs of lid-sutured monkeys. In all deprived animals, the cross-sectional areas of deprived puffs decreased most rapidly in the upper layers (2 and 3A). In long-term enucleated (60 wks) and TTX-treated (4 wks) monkeys, puff area was severely reduced in layer 3B, while reactivity in layer 3B appeared partially spared in lid-sutured monkeys. The density of the CO reaction product was significantly and evenly reduced throughout deprived puffs for all of the monkeys examined; however, this decrease was less severe in adult monkeys lid-sutured for 11 wks. Although no evidence for cell loss was obtained, all three forms of visual deprivation led to lower counts of neuronal perikarya with high levels of CO reaction product in both deprived puff and interpuff areas. This effect was less marked in the deprived puffs of monkeys lid-sutured for 2.5 and 3 yrs, suggesting recovery of CO activity in some neurons. Neurons in deprived puffs and interpuffs were generally similar in size to those in nondeprived regions, although CO-reactive cells were significantly smaller in the deprived puffs of monkeys enucleated for 28.5 or 60 wks. These results indicate that the metabolic response of neuronal elements in supragranular striate cortex depends upon the nature of the visual deficit. The partial sparing of CO reactivity in deprived puffs of lid-sutured monkeys may reflect the continued transmission of certain types of visual stimuli through a closed eyelid.

Much attention has been paid to the effect of various types of neonatally induced retinal insults on neurons of the cat lateral geniculate nucleus (LGN). Little is known about cellular adjustment to functional altertions commencing in the adult. The present study was aimed at examining the effect of monocular enucleation or retinal impulse blockade on mature neurons of the cat LGN and perigeniculate nucleus. In addition to labeling the relay neurons with cytochrome oxidase (CO) histochemistry and immunohistochemistry, and presumed interneurons with GABA immunohistochemistry, the two markers were also combined in the same section. The results showed that GABA-immunoreactive neurons in the LGN can be subdivided into two major groups: highly immunoreactive neurons (Hir) and moderately immunoreactive neurons (Mir). These two groups differed slightly in their size and CO levels. With monocular enucleation or TTX treatment, there was a reduction in the numerical density of Hir and a concomitant increase in Mir in the affected laminae. However, there was no evidence of a reduction in GABA immunoreactivity in neurons of the perigeniculate nucleus. With regard to relay cells our data were in agreement with our previous findings (Kageyama & Wong-Riley, 1985) that there was a statistically significant positive correlation between cell size and CO levels, so that neurons with large cross-sectional areas were predominately darkly reactive for CO (Dco), while medium-to-small neurons were mainly moderate to lightly reactive (M/Lco). After monocular enucleation or TTX injections, the numerical density and size of Dco were significantly reduced, while those of M/Lco were proportionally increased in the affected laminae, indicating a conversion of some Dco to M/Lco. The results indicate that the maintenance of CO levels in predominately the large relay cells and GABA levels in a subclass of small neurons of the mature cat LGN are activity-dependent and are highly sensitive to retinal insults.

The telencephalic target of the thalamofugal visual pathway in birds, the visual wulst, is part of the hyperstriatum accessorium/dorsale in the bird's brain. In this study, we tried to determine the exact location of the visually responsive area in the zebra finch by recording visually evoked potentials (VEPs) from different sites throughout the hyperstriatum and calculating current source densities (CSDs). In addition, we examined the influence of ipsilateral and contralateral stimuli on stimulus processing within this area, and tried to get insight into the neuronal machinery of the thalamofugal pathway by application of drugs such as tetrodotoxin (TTX) and picrotoxin.

About two-thirds of the hyperstriatum is responsive to contralateral stimuli but only a small portion responds to ipsilateral stimuli. Contralateral visual information arrives in the hyperstriatum dorsale (HD) and is processed further to the hyperstriatum accessorium (HA).

The small influence of ipsilaterally evoked potentials is not due to inhibition by the activity of the contralateral eye, as could be demonstrated previously for the ectostriatum. Instead, our results show that ipsilaterally evoked potentials are inhibited at least in part by a projection from the contralateral visual wulst.

Unilateral retinal impulse blockage with tetrodotoxin (TTX) induces reversible shrinkage and decreased cytochrome oxidase (CO) activity in alternate rows of supragranular, CO-rich puffs in the adult macaque striate cortex (Wong-Riley & Carroll, 1984b: Carroll & Wong-Riley, 1987). The present study extended the findings to the electron-microscopic (EM) level to determine if various neuropil profiles in control puffs exhibit heterogeneous levels of CO activity, and whether specific processes were more susceptible to intravitreal TTX than others.

Within the neuropil of control puffs, 60% of the total mitochondrial population resided in dendrites, and the majority of dendritic mitochondria were highly reactive for CO. Axon terminals forming symmetrical synapses also contained darkly reactive mitochondria, whereas those forming asymmetrical synapses possessed very few and mainly lightly reactive mitochondria. Unmyelinated axon trunks, myelinated axons, and glia all exhibited low levels of CO activity. Synaptic count revealed a 3:1 ratio of asymmetrical to symmetrical synapses.

Intravitreal TTX for 2–4 weeks adversely affects dendrites and symmetrical terminals much more so than other neuropil processes. There was a general decrease in darkly and moderately reactive mitochondria and an increase in lightly reactive mitochondria throughout the puffs, especially in dendrites. This indicates that afferent blockade is more detrimental to processes of higher metabolic activity. Changes also differed between central and peripheral regions of puffs, and indications of axonal and synaptic reorganization were more evident in the latter. Thus, stabilization of neuronal structure and synapses appears to be activity-dependent even in the adult. A working model of these metabolic and morphological responses to chronic TTX is proposed.

Our previous light-microscopic study indicates that unilateral retinal impulse blockage with tetrodotoxin (TTX) causes a reversible decrease of cytochrome oxidase (CO) in alternating rows of metabolically active zones (puffs) in the adult macaque striate cortex (Wong-Riley & Carroll, 1984b). The goal of the present study was to determine if TTX blockade adversely affects all neurons or only a subpopulation of neurons within the puffs. Three major neuronal types were identified based on mitochondrial CO activities and morphological characteristics. Type A neurons were the most prevalent, consisting of small pyramidal and nonpyramidal neurons that received only symmetrical axosomatic synapses. They had little cytoplasm and relatively low levels of CO activity, and showed the least change with TTX treatment. Type B cells were medium-to-large pyramidal neurons that received exclusively symmetrical axosomatic synapses and were moderately reactive for CO. Impulse blockage caused a decrease in mitochondrial size and packing density, but somal size remained within the control range. Type C cells were medium-sized nonpyramidal neurons contacted by both asymmetrical and symmetrical axosomatic synapses. They contained abundant darkly reactive mitochondria and presumably are metabolically the most active. This cell type suffered the greatest decrease in somal size and packing density of mitochondria, particularly the darkly reactive ones. A rare fourth cell type, type D, was a small, darkly reactive nonpyramidal variety that gave rise to somatodendritic synapses. Their low occurrence prevented statistical analysis under normal and TTX-treated conditions. These data indicate that retinal impulse blockade is most detrimental to the metabolically most active neurons in the adult primate cortical puffs. The alterations are not permanent, because the effects of TTX are fully reversible (Carroll & Wong-Riley, 1987).

To determine whether central communication of retinal signals is necessary for the development of an experimentally induced myopia, tree shrews were exposed to monocular deprivation (MD) while the action potentials of retinal cells in the deprived eye were blocked with intravitreally injected tetrodotoxin (TTX-MD animals). TTX injections (0.6 μ 3 μL) and MD began about 15 days after eye opening, at the start of the susceptible period for the development of lid-suture myopia. Six injections were given, one every second day to produce 12 days of MD and TTX-blockade. Control TTX animals (TTX-open) received TTX in one eye, but not MD, on the same injection schedule and were always found to be behaviorally unresponsive to visual stimuli through the injected eye indicating that TTX blocked central communication of action potentials. Other control animals received intravitreally injected saline in either an open eye (saline-open), or an MD eye (saline-MD). A sham-injected group (sham-inj-MD) received MD and all anesthetic and surgical manipulations except for penetration of the sclera. In all groups, one eye in each animal was an untreated control.

Two effects were found. All MD groups, including the TTX-MD animals, developed a significant vitreous chamber elongation in the deprived eye, indicating that an experimental myopia developed despite ganglion cell blockade. Thus, retinal mechanisms in tree shrew can detect the presence of a degraded visual image and produce an experimental myopia that does not depend on the receipt of visual messages by central neural structures. In addition, eyes in which the sclera was punctured had smaller vitreous chamber depths than comparable uninjected eyes, indicating that puncturing the sclera reduced the normal elongation. These data suggest that forces within the eye normally contribute to its expansion and may be resisted by the choroid and/or the sclera.

Glutamate and its various receptors are known to play an important role in excitatory synaptic transmission throughout the CNS, including the primary visual cortex. Among subunits of the AMPA receptors (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), subunit 2 (GluR2) is of special significance because it controls their Ca2+ permeability. In the past, this subunit has been studied mostly in conjunction with other AMPA subunits. The present study sought to determine if GluR2 alone has a distinct laminar distribution in the normal macaque visual cortex, and if its pattern correlated with that of cytochrome oxidase (CO) under normal and monocularly deprived conditions. In the normal adult cortex, GluR2 immunoreactivity (ir) had a patchy distribution in layers II/III, in register with CO-rich puffs. GluR2-ir highlighted the upper border of layer II, the lower border of layer IV (previously termed IVCβdark) and, most prominently, layer VI. Labeled neurons were primarily of the pyramidal type present in the upper border and lower half of layer VI, layers II/III, and scattered in layers V and upper IVB. Labeled nonpyramidal cells were large in layer IVB and small in IVCβdark. Notably, the bulk of CO-rich layers IVC and IVA had very low levels of GluR2-ir. At fetal day 13, however, GluR2 labeling showed a honeycomb-like pattern in layer IVA not found in the adult. A fragment of GluR2 cDNA was generated from a human cDNA library, and in situ hybridization revealed an expression pattern similar to that of GluR2 proteins. After 1–4 weeks of monocular impulse blockade with tetrodotoxin (TTX), alternating rows of strong and weak GluR2-ir in layers VI and II/III appeared in register with CO-labeled dark and light ocular dominance columns in layer IVC and puffs in II/III, respectively. Our results indicate that various cortical layers are differentially influenced by glutamate. The bulk of the major geniculate-recipient layers IVC and IVA have low levels of GluR2, presumably favoring synaptic transmission via Ca2+-permeable glutamate receptors. GluR2 plays a more important role in supragranular and infragranular layers, where the initial geniculate signals are further modified and are transmitted to other cortical and subcortical centers. The maintenance of GluR2 in these output layers is governed by visual input and neuronal activity, as monocular impulse blockade induced a down-regulation of this subunit in deprived ocular dominance columns.

During early mammalian development, inputs from the two retinas intermix within the lateral geniculate nucleus (LGN), then segregate during the first postnatal week into layers that receive input from a single retina. Functionally, the LGN also changes markedly during the first postnatal month; early geniculate responses to retinal input are mainly excitatory, then inhibitory circuits mature within the LGN. These remarkable changes in form and function of the retinogeniculate pathway occur at a time when patterned visual activity is not present, but retinal ganglion cells already manifest spontaneous action potential activity. To examine the role of early retinal activity in these critical developmental processes, we placed the slow release polymer Elvax embedded with tetrodotoxin (TTX) into the vitreous chamber of one or both eyes of neonatal ferrets. Animals receiving monocular injection of TTX had the other eye treated with Elvax containing control citrate buffer. Intraocular injection of horseradish peroxidase was made at the end of the period of TTX treatment to reveal the retinal terminals in the LGN. Chronic monocular or binocular blockade of retinal activity during the first postnatal week did not prevent eye-specific segregation, although it made the boundaries between layers less distinct. Retinal terminals ended preferentially in the appropriate layer, but a large number of terminals were also present in the inappropriate layer. Further segregation was achieved during the second postnatal week of activity blockade, when most retinal terminals ended preferentially in the appropriate geniculate layer and sharper layer boundaries were present. However, a small but significant number of terminals still extended into the inappropriate layer. Together, these findings indicate that monocular as well as binocular blockade of retinal activity resulted in some anomalous retinogeniculate projections and delayed eye-specific patterning, but segregation was largely intact at the end of the second postnatal week. We also report here that intraocular tetrodotoxin had a marked effect on the maturation of intrinsic geniculate circuits prior to eye opening. Whole-cell patch-clamp recordings in the LGN slice preparation revealed that activity blockade prevented the maturation of the slow, but not the fast, hyperpolarizing potential of LGN neurons during the first postnatal month and up to P38, the oldest age studied. In conclusion, these results indicate that spontaneous retinal activity modulates the time course of binocular segregation but does not alone account for the segregation of retinogeniculate terminals. However, early retinal activity plays an important role in developing the intrinsic circuitry of the LGN.

Patch-clamp recordings were made from ganglion cells in an in vitro dark-adapted rabbit retina preparation. Cells were stimulated by images generated on a computer monitor and focussed onto the photoreceptors. Excitatory inward currents were recorded in response to spot stimuli centered on the somas of the recorded cells. Center illumination of on-brisk-transient cells produced large transient excitatory postsynaptic currents (EPSCs) which were invariably followed by a small steady-state inward component. Illumination of a centered annulus failed to elicit the transient EPSC. Simultaneous illumination of the annulus and the center spot blocked the large transient EPSC, consistent with activation of an inhibitory surround. Application of tetrodotoxin (TTX), which blocks sodium-dependent action potentials, also blocked the surround inhibition in ON-brisk transient cells as well as some other classes of ganglion cells. It is concluded that, in some ganglion cell classes, the surround is generated largely through the activity of spiking neurons, and it is suggested that the amacrine cells in the inner plexiform layer are involved.

The neuromuscular sodium currents of early invertebrates such as platyhelminths display distinctive kinetic and pharmacological properties. We have cloned a cDNA from the horseshoe crab flatworm Bdelloura candida that encodes a protein homologous to the primary subunit of voltage-gated sodium channels. The B. candida protein, named BdNa1, exhibits amino acid identity of 40–47% to sodium channels of vertebrates and higher invertebrates. BdNa1 has the multidomain structure characteristic of sodium channels, and is most highly conserved in the hydrophobic transmembrane segments and the regions that form the pore of the channel. Northern blot analysis confirms the presence of a 5·4 kb BdNa1 transcript in B. candida tissue. The information provided by analysis of the BdNa1 sequence offers insight into the physiology of platyhelminth sodium currents.