Relative number of cells projecting from contralateral and ipsilateral nucleus isthmi to loci in the optic tectum is dependent on visuotopic location: horseradish peroxidase study in the leopard frog.

The leopard frog optic tectum is the principal target of the contralateral retina. The retinal terminals form a topographic map of the visual field. The tectum also receives bilateral topographic input from a midbrain structure called nucleus isthmi. In this study we determined the relative strength of n. isthmi projections to different loci in the tectum. Horseradish peroxidase (HRP) was applied at single superficial tectal locations in a series of leopard frogs. The application sites were distributed across the tectum. Retrogradely filled cells were counted in ipsilateral and contralateral nucleus isthmi. Although all regions of the tectum receive input from both n. isthmi, the relative number of labeled cells in the two n. isthmi is dependent on visuotopic location. Input to the rostromedial tectum representing the visual field ipsilateral to the labeled tectum comes primarily from the contralateral n. isthmi. Input to the caudolateral tectum representing the visual field contralateral to the labeled tectum originates mostly from the ipsilateral n. isthmi. Tectal application sites representing the visual midline had approximately equal numbers of labeled cells in the two n. isthmi. The results are similar at postapplication survival times ranging from 2 to 14 days. Using application of HRP to rostral tectum and application of nuclear yellow to caudal tectum, we show that the anisotropy in isthmi labeling is not due to take up of these labels by isthmotectal fibers passing through the application sites that terminate elsewhere.

Prey selection in the leopard frog: choosing in biased and unbiased situations.

When given the option between a ground-level prey presented in front and another prey presented 90 degrees to the side, leopard frogs have a front preference. When given a choice between prey objects presented at the left and right sides, some individual leopard frogs have a side preference. Repeated prey object presentations at one side predispose leopard frogs to respond to the opposite side when presented with prey objects at both sides. The phenomenon is preserved through a half minute delay between single prey presentations (biasing) and two prey presentations (testing) but not through a three-minute delay between biasing and testing. Ten biasing presentations on a side are sufficient to induce opposite side preference, while three biasing presentations are insufficient. Attempts to permanently modify preferences by completely exhausting responsiveness to a single side were unsuccessful. A neural model for the effect of biasing on behavior is shown.

Calcium signals monitored from leopard frog optic tectum after the optic nerve has been selectively loaded with calcium sensitive dye.

We loaded adult leopard frog optic nerves with the calcium-sensitive dye Calcium Green-1 3000 mw dextran conjugate. The dye was transported to the optic tectum in approximately 6 days and selectively labeled optic nerve terminals as seen with confocal microscopy. Viewed with an intensified CCD system, electrical stimulation of the optic nerve in vitro increases Calcium Green-1 fluorescence significantly. With increasing number of pulses in pulse trains there was increased presynaptic facilitation as measured by increased fluorescence. Addition of nicotine to the bathing solution increased baseline fluorescence. These results suggest that Calcium Green-1 dextran conjugate can be actively transported in adult nerve fibers over a significant distance and is retained in presynaptic terminals in a form that allows monitoring of presynaptic calcium levels.

Seeing beyond the midline: the role of the contralateral isthmotectal projection in the leopard frog.

The ground level visual field of each eye of the leopard frog includes the entire ipsilateral 180-deg field and approximately 60 deg into the frontal contralateral field. When one eye is covered with an opaque patch, a frog responds to prey stimuli over the entire field of the other eye. Nevertheless, when one optic nerve is cut, the animal responds to prey in the ipsilateral hemifield of the connected eye, but only responds as far as about 30 deg past the frontal midline. If one optic tract is cut, the animal does not respond to prey past the frontal midline. We hypothesized that the responses past the frontal midline might be mediated by input from contralaterally projecting isthmotectal fibers. These fibers originate in the nucleus isthmi, a posterior midbrain structure. We found that when we placed an opaque patch over one eye and either ablated the ipsilateral nucleus isthmi, or cut crossing isthmotectal fibers in the optic chiasm, or blocked input to nucleus isthmi by ablating the ipsilateral tectal lobe, animals did not respond to prey stimuli past the frontal midline. We found that when we placed an opaque patch over one eye and cut crossing optic fibers in the anterior part of the optic chiasm (sparing crossing isthmotectal fibers), animals responded to prey stimuli in the nasal half of the seeing eye's contralateral frontal field. Our results suggest that contralaterally projecting isthmotectal fibers enable the frog to respond to stimuli past the frontal midline. We suggest a one-dimensional model of how nucleus isthmi influences tectal function.

Regrowth of optic fibers and behavioral recovery after optic chiasm transection.

We studied the effect of optic chiasm midline transection on visually guided behavior and retinotectal fiber regrowth in frogs. After complete transection, frogs do not respond to visually presented prey and looming stimuli. Beginning about 2 months later there is recovery of visual function. However, unlike recovery after optic nerve transection, animals respond as if the stimulus were not at its actual position, but at the symmetric position in the contralateral field. For instance, if a prey stimulus is located 5 cm away from the recovered frog at an eccentricity of 40 degrees to the left of the midline, the animal will respond as if the stimulus were 5 cm away at 40 degrees right. Further, these animals typically respond to looming stimuli not by jumping away from the stimulus, but by either colliding with the stimulus or jumping toward the side from which the stimulus approaches. These behaviors persist throughout the testing period, up to 17.5 months postlesion. Electrophysiological recordings reveal that visual activity in the optic tectum is retinotopically organized but driven primarily by stimuli to the ipsilateral eye. HRP histochemistry reveals that some regenerated retinal fibers are found to cross at the midline of the chiasm. Thus, the midline is not impenetrable to crossing retinal fibers. Frogs with cut of 3/4 of the chiasm respond normally to prey stimuli initially but later respond as if the stimuli are at mirror image locations. In these animals most retinotectal fibers project to the ipsilateral tectum despite the presence of intact contralaterally projecting retinotectal fibers during the recovery period.

Studies on the optic chiasm of the leopard frog. I. Selective loss of visually elicited avoidance behavior after optic chiasm hemisection.

We hemisected either the posterior or anterior portion of the optic chiasm and found that frogs were unresponsive to large looming stimuli anywhere in the visual field. Nonetheless, the animals responded to prey stimuli throughout the visual field. Responses to looming stimuli returned in 1 to 8 weeks post-surgery. After complete transection of the chiasm animals were unresponsive to both prey and large looming stimuli. Frogs responded normally to prey and looming stimuli if less than half the optic chiasm was cut or if the postoptic commissure was cut. Since responses to looming stimuli returned before cut optic fibers could regenerate, these results suggest that visual information concerning prey and large looming objects are mediated by separate optic nerve fiber systems.

Studies on the optic chiasm of the leopard frog. II. Organization of retinotectal fibers in the optic chiasm.

The organization of retinotectal fibers in the optic chiasm was investigated using horseradish peroxidase (HRP) histochemistry and electrophysiological recording. HRP injection into a small region of the tectum led to retrograde staining of labeled fibers in a circumscribed region of the chiasm and staining of labeled ganglion cells in the contralateral retina. In each instance labeled tissue was spread over a greater proportion of the area of a chiasm section than over the flattened retina. Fibers originating in central (older) retina are located in dorsal chiasm. Fibers originating in peripheral (younger) retina are located in ventral chiasm. Viewed with the electron microscope, labeled unmyelinated fibers are admixed with labeled myelinated fibers. Neuronal activity was monitored with an extracellular microelectrode from points in dorsoventral tracks in the chiasm. Multiple units were recorded at each chiasm location. Using visual stimuli, the receptive fields of the units were mapped. The fields were distributed along an arc across the visual field. At ventral chiasm recording sites the arc was in the peripheral part of the visual field. In succeeding dorsal sites the arcs were concentrically arranged so that the more dorsal the chiasm recording site, the more central was the arc in the visual field. Thus, in the optic chiasm, retinal fibers appear to be organized chronotopically but not retinotopically. Fibers of the same age but from different locations in the retina are mixed together.

Synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi in Rana pipiens.

The nucleus isthmi is reciprocally connected to the ipsilateral optic tectum. Ablation of the nucleus isthmi compromises visually guided behavior that is mediated by the tectum. In this paper, horseradish peroxidase (HRP) histochemistry and electron microscopy were used to explore the synaptic interrelationships between the optic tectum and the ipsilateral nucleus isthmi. After localized injections of HRP into the optic tectum, there are retrogradely labeled isthmotectal neurons and orthogradely labeled fibers and terminals in the ipsilateral nucleus isthmi. These terminals contain round, clear vesicles of medium diameter (40-52 nm). These terminals make synaptic contact with dendrites of nucleus isthmi cells. Almost half of these postsynaptic dendrites are retrogradely labeled, indicating that there are monosynaptic tectoisthmotectal connections. Localized HRP injection into the nucleus isthmi labels terminals primarily in tectal layers B, E, F, and 8. The terminals contain medium-sized clear vesicles and they form synaptic contacts with tectal dendrites. There are no instances of labeled isthmotectal terminals contacting labeled dendrites. Retrogradely labeled tectoisthmal neurons are contacted by unlabeled terminals containing medium-sized and small clear vesicles. Fifty-four percent of the labeled fibers connecting the nucleus isthmi and ipsilateral tectum are myelinated fibers (average diameter approximately 0.6 microns). The remainder are unmyelinated fibers (average diameter approximately 0.4 microns).

Behavioral and physiological consequences of unilateral ablation of the nucleus isthmi in the leopard frog.

Unilateral lesions of the nucleus isthmi result in a scotoma to visually presented prey and threat stimuli in the contralateral monocular visual field. There is a correlation between the size of the scotoma and the amount of n. isthmi ablated. Following the lesion, there is a regression of the scotoma in the nasal part of the visual field which then stabilizes. Upon longer behavioral examination, the animals can be divided into two classes: (1) animals in which the scotoma remains relatively stable in size for up to two years, and (2) animals which recover from the scotoma. In the latter group, there tends to be damage to both the n. isthmi and the posterodorsal tegmental nucleus which lies mediocaudal to the n. isthmi. Electrophysiological recording from positions within the area of the optic tectum representing the scotoma reveal an average threefold increase in the size of the multiunit receptive fields compared to mirror image positions in the contralateral optic tectum.

Nucleus isthmi: its contribution to tectal acetylcholinesterase and choline acetyltransferase in the frog Rana pipiens.

The distribution of acetylcholinesterase and the activity of choline acetyltransferase was studied in the tecta of normal frogs and frogs without retinal and/or nucleus (n.) isthmi inputs. In normal animals acetylcholinesterase activity is found primarily in three bands in the outer layers of the tectum-lamina A, laminae C-F, and lamina G. After retinal and contralateral n. isthmi deafferentation three distinct bands of tectal acetylcholinesterase activity are still present. After bilateral n. isthmi deafferentation there is loss of activity in lamina G and reduced activity in lamina A. With retinal and ipsilateral n. isthmi deafferentation, activity is seen only in lamina A. With retinal and bilateral n. isthmi deafferentation there is virtually no acetylcholinesterase activity in the outer tectal layers. Following unilateral retinal deafferentation there is no statistically significant difference in choline acetyltransferase specific activity between intact and deafferented tectal lobes after two, four and nine weeks. With unilateral nucleus isthmi lesions and survival times of between 10 and 40 days, choline acetyltransferase specific activity in the tectal lobe ipsilateral to the ablation is approximately 38% of the specific activity of the contralateral lobe. With bilateral n. isthmi lesions there is a strong correlation between amount of n. isthmi ablated and reduction of choline acetyltransferase activity. In vitro tectal acetylcholine synthesis was also determined in animals with unilateral n. isthmi ablation. On average, tectal lobes ipsilateral to the ablated n. isthmi synthesize acetylcholine at a rate which is approximately 58% of that of contralateral tecta. Collectively, these results imply that n. isthmi is the sole cholinergic input to the frog optic tectum, with ipsilaterally projecting isthmotectal fibers accounting for the greater share.

Relationship between isthmotectal fibers and other tectopetal systems in the leopard frog.

We studied the relationship of isthmotectal input to other tectal afferent fiber systems in three ways. 1) Using horseradish peroxidase (HRP) histochemistry, we determined the nonretinal inputs to the superficial tectum. In different sets of animals we a) applied HRP to the tectal surface; b) inserted HRP crystals into the tectum; c) injected small volumes of HRP solutions into the superficial tectum. N. isthmi accounts for more than 65% of the nonretinal extrinsic input in the superficial tectal layers. One set of fibers from the contralateral n. isthmi projects to the most superficial layer. Fibers from posterior thalamus and tegmentum project to both superficial and deeper layers in the tectum, but not to the most superficial layer. The ipsilaterally projecting isthmotectal fibers terminate in the deeper superficial layers. 2) We investigated the relationship between retinofugal and contralaterally projecting isthmotectal pathways. We orthogradely labelled n. isthmi fibers by unilateral HRP injections into n. isthmi, and we also labelled retinal fibers by injecting tritiated l-proline into both eyes. In such animals contralaterally projecting isthmotectal fibers cross in the dorsal posterior region of the optic chiasm. From the chiasm to the tectum isthmotectal fibers and retinofugal fibers are admixed. 3) We determined whether other fiber systems cross with contralaterally projecting isthmotectal fibers. We cut the posterior part of the optic chiasm and applied HRP crystals to the cut. Only n. isthmi and retina are retrogradely labelled.

Cholinergic innervation of the optic tectum in the frog Rana pipiens.

An immunohistochemical method for choline acetyltransferase (ChAT) identifies presumably cholinergic axons in two retino-receptive laminae in the optic tectum of the frog Rana pipiens. Following eye enucleation there is no loss of immunoreactive axons in the optic tectum. Following unilateral ablation of the nucleus isthmi there is a near-total loss of ChAT-positive axons in the superficial cholinergic lamina contralaterally and in the deeper cholinergic lamina ipsilaterally. Thus, the cholinergic innervation of the tectum appears to derive from the nucleus isthmi. However, ChAT-positive staining of the basal optic nucleus does depend upon an intact retinal input and could derive from either retinal axons or some system trophically dependent on them.

Nucleus isthmi provides most tectal choline acetyltransferase in the frog Rana pipiens.

Up to 9 weeks following the removal of unilateral retinal input, choline acetyltransferase (ChAT) activity in the de-afferented tectal lobe is not significantly different from the intact tectal lobe. At 14 weeks, there is a 29% increase in the de-afferented side compared to the intact side. Following unilateral lesion of nucleus isthmi, ChAT activity in the tectal lobe ipsilateral to the lesion is approximately 30% of that measured in the contralateral lobe. Following bilateral n. isthmi lesion, ChAT activity in each tectal lobe is reduced by approximately 94% from intact tectal lobe controls. Thus, nucleus isthmi is the principal source of cholinergic input to the tectum.

Ablation of nucleus isthmi leads to loss of specific visually elicited behaviors in the frog Rana pipiens.

Ablation of the frog's nucleus isthmi results in a visual scotoma contralateral to the lesion. Within the scotoma, animals do not respond to visually presented prey or threats. The locus of visual loss is related to the area of isthmal tissue ablated. With complete unilateral ablation, a frog displays no visually elicited prey-catching or threat-avoidance behaviors in the entire monocular field.

2-Deoxyglucose labelling of the infrared sensory system in the rattlesnake, Crotalus viridis.

Infrared (IR) responsive nuclei in the rattlesnake Crotalus viridis were identified by using 14C-2-deoxyglucose (2DG) and autoradiography. Following 2DG intracardial injection, the IR-sensitive pit organ was stimulated periodically with an IR stimulus for 5 hours. The nucleus of the lateral descending trigeminal tract (LTTD, the primary IR sensory nucleus) was labelled heavily with 2DG. Labelling was bilateral, but somewhat heavier ipsilateral to the stimulated pit organ. The nucleus reticularis caloris (RC, the secondary nucleus of the IR system) was lightly labelled ipsilaterally. The middle laminae of the contralateral optic tectum (which contain IR-responsive units) were distinctly labelled; the corresponding layers of the ipsilateral tectum were lightly labelled. A subcerebellar nucleus not known to be part of the IR system was heavily labelled bilaterally. No consistent labelling was found in the diencephalon or telencephalon. Since units in the LTTD do not respond to stimulation of the contralateral pit yet the LTTD is labelled with 2DG when there is contralateral pit stimulation, several controls were carried out. Unilateral injection of 3H-proline into LTTD revealed no projection to the contralateral LTTD. In a monocularly, visually stimulated animal with both pits occluded, the LTTD still showed heavy but equal 2DG labelling bilaterally. In addition, the outer layers of the contralateral optic tectum were heavily labelled. No 2DG labelling of the LTTD was obtained when branches of the trigeminal nerve innervating the LTTD were previously cut. These results suggest that much of the 2DG labelling in the LTTD is due to spontaneous ongoing activity from the pit organ rather than from IR evoked activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Basal optic complex in the frog (Rana pipiens): a physiological and HRP study.

The basal optic projection in the frog Rana pipiens has been investigated by single-unit extracellular recording and horseradish peroxidase (HRP) histochemistry. We approached the projection from the ventral side of the brain and recorded single units in the basal optic projection proper as well as in the adjacent dorsomedial region (jointly called the basal optic complex). We found a) units responsive to stimuli moving in a vertical direction, b) an approximately equal number of units responsive to stimuli moving in a horizontal direction, and c) a smaller number of units responsive to changes in ambient light and moving stimuli without direction selectivity. Directional units display significant maintained activity and usually decrease their firing rate in response to stimulus motion in a direction opposite to that which elicits the maximal increase in firing rate. Receptive-field sizes for directional units ranged from 10 to 60 degrees. All units displayed vigorous excitatory response to a wide variety of moving stimuli within the velocity range of 0.2-10 degree/s. HRP histochemistry shows that in addition to the retina, the basal optic complex is connected to three principal areas: the ipsilateral tegmental griseum centrale, the ipsilateral dorsal ventrolateral nucleus of the anterior thalamus, and the ipsilateral posterior thalamic nucleus. In addition, a pathway was observed consisting of two groups of cells that send axons to the ipsilateral rostroventral medulla. This pathway originates a) in cells whose somata lie within the dorsomedial aspect of the basal optic complex (BOC); and b) in cells whose somata lie immediately outside the BOC in the adjacent gray, with apical dendrites extending into the BOC. Some of these fibers continue to the level of the spinal cord. Injection of HRP into the rostroventral medulla led to retrograde labeling of cells of the BOC.

Removal of retinal input fails to elicit isthmo-tectal sprouting in the frog.

The retina and nucleus isthmi both project in laminar fashion to the superficial layers of the frog's tectum. In order to determine whether isthmo-tectal axons show collateral sprouting after retinotectal input is removed, we injected [3H] proline into the nucleus isthmi and measured the volume of the crossed isthmo-tectal projection. We found no evidence of collateral sprouting.

Anatomy and physiology of a binocular system in the frog Rana pipiens.

The locations of tectal neurons projecting to nucleus isthmi (n. isthmi) were found by iontophoretic injection of horseradish peroxidase (HRP) into n. isthmi. After retrograde transport, stained tectal somata are found to lie almost exclusively in layer 6 and below of the ipsilateral tectum. Many cells are colored throughout the extent of their dendrites into the fine rami, giving the appearance of a Golgi stain. Nucleus isthmi receives projections from the ipsilateral tectum and from no other region. Nucleus isthmi units recorded electrically respond to visual stimuli and are arranged in a topographic map of the visual field. There are two types of receptive fields, those with small centers and those with large centers. The small centers are about 3-5 degrees in diameter, similar to type 2 optic nerve fibers. Their response is to many of the same geometric features of stimulus as excite type 2 fibers. The large centers are at least 7-10 degrees in diameter and respond to many of the same features as excite types 3 and 4 optic nerve fibers. The responsiveness of small and large center n. isthmi units is very similar to the elements of the ipsilateral visual field projection onto tectum, i.e. the neuropilar units recorded in layers A and 8 of the tectum when the contralateral eye is occluded. These are in strong contrast to those of tectal cells of layer 6 and below, which have large receptive fields, show far less vivacious response, adapt extremely rapidly to repeated stimuli and are hard to describe in terms of characteristic stimuli because they are unresponsive most of the time. We suggest, therefore, that the axons of tecto-isthmic cells are quite active and that their cell bodies, located in layer 6 and below, only fire occasionally on the firing of their axons.

The infrared trigemino-tectal pathway in the rattlesnake and in the python.

We have studied the infrared trigemino-tectal pathway of the rattlesnake (Crotalus viridis) and the python (P. reticulatus). In the rattlesnake, horseradish perosidase (HRP) injections into the nucleus reticularis caloris (RC) result in retrograde filling of cells in the ipsilateral nucleus of the lateral descending trigeminal tract (LTTD) and in the anterograde labelling of terminal fields in the contralateral optic tectum, confirming our previous finding of an RC-tectal projection. The primary projection of the pit organ of the rattlesnake was traced by injecting cobalt chloride into the pit, demonstrating that the pit organ projects exclusively to the ipsilateral LTTD. Electrophysiological recording from single units in the RC shows that these cells respond to infrared stimulation. Taken together, these results demonstrate that the infrared pathway in the rattlesnake proceeds from the pit organ to the LTTD, to the RC, to the contralateral tectum. In contrast, HRP injection into the tectum of the python results in the retrograde filling of the large cells of the contralateral LTTD. Thus, a direct LTTD-tectal projection occurs in the python. The cells of the rattlesnake RC and the larger cells of the python LTTD stain heavily for acetylcholinesterase activity and have a similar multipolar appearance, suggesting that the tectal-projecting cells in the two species may have a common origin.

Connections of the tectum of the rattlesnake Crotalus viridis: an HRP study.

We have studied the connections of the tectum of the rattlesnake by tectal application of horseradish peroxidase. The tectum receives bilateral input from nucleus lentiformis mesencephali, posterolateral tegmental nuclei, anterior tegmental nuclei and periventricular nuclei; ipsilateral input from nucleus geniculatus pretectalis, and lateral geniculate nucleus pars dorsalis; and contralateral input from dorso-lateral posterior tegmental nucleus and the previously undescribed nucleus reticularis caloris (RC). RC is located on the ventro-lateral surface of the medulla and consists of large cells 25--45 micrometer in diameter. Efferent projections from the tectum can be traced to the ipsilateral nucleus lentiformis mesencephali, the ipsilateral lateral geniculate region, anterior tegmental region and a wide bilateral area of the neuropil of the ventral tegmentum and ventral medualla. We have not found any direct tectal projections from the sensory trigeminal nuclei including the nucleus of the lateral descending trigeminal tract (LTTD). We suggest that in the rattlesnake, RC is the intermediate link connecting LTTD to the tectum.