Hermes H. Yeh

Photic regulation of c-fos expression in neural components governing the entrainment of circadian rhythms

The rapid and transient induction of the proto-oncogene c-fos in mature neurons within the brain occurs in response to a variety of extracellular stimuli. To determine whether lighting conditions influence c-fos gene expression in the primary neural structures mediating the photoentrainment and generation of mammalian circadian rhythms, the expression of the c-fos protein (Fos) and related proteins in the retina and suprachiasmatic nuclei (SCN) of the anterior hypothalamus was examined immunohistochemically in rats exposed to a light-dark cycle of 12 h of light and 12 h of darkness (LD 12:12), constant light (LL), or constant dark (DD). The retina exhibited clear light-dark differences in the expression of Fos protein(s), such that immunopositive nuclei were readily evident during exposure to light (i.e., during the day of diurnal lighting or in LL), but were absent during exposure to darkness. In the SCN, the distribution of Fos immunoreactivity within specific subfields was differentially affected by photic conditions. Following exposure to light, a dense population of Fos-immunopositive cells was found in close association with the immunohistochemically distinct cell and fiber populations distinguishing the ventrolateral subfield of the SCN. In dark-exposed animals, Fos-immuno-reactive profiles were distributed throughout the SCN in areas coextensive with the immunohistochemical localization of peptidergic neural elements in both the ventrolateral and dorsomedial subfields. As a consequence of this light-dark difference in the distribution of Fos immunoreactivity, the density of labeled cells was increased within the ventrolateral SCN, but was decreased within the dorsomedial subfield, as a result of exposure to light versus darkness.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Kinase C-like immunoreactivity in rod bipolar cells of the rat retina: A developmental study

In the retina of a variety of vertebrate species, a monoclonal antibody against protein kinase C (PKC) has been shown to label preferentially bipolar cells. Although the functional consequences of PKC activation in these cells is yet to be revealed, the present study was motivated in part by the possibility that the antibody might be used as a selective marker for examining the development of bipolar cells in the rat retina. Here, the developmental pattern and the dynamic changes of retinal cells expressing PKC-like immunoreactivity (PKC-LI) were studied and analyzed throughout postnatal life until adulthood. Upon its initial detection by immunohistochemistry on postnatal day (PD)-10, faint PKC-LI was limited to the central region of the retina, labeling cell bodies located at the scleral margin of the inner nuclear layer (INL) adjacent to the outer plexiform layer (OPL). On subsequent days, PKC-LI spread progressively to the peripheral retina and axon terminal bulbs at the vitreal margin of the inner plexiform layer (IPL) began showing the first signs of immunoreactive labeling. Not until PD-15, the time of eye opening, did PKC-LI in these cells increase to the extent such that their thin axons were immunoreactive. Each of these axons traversed the entire thickness of the IPL and divided into two or three short branches before ending as enlarged terminal bulbs. The morphology and the location of PKC-LI cells in both the developing and adult retina observed in our study are consistent with them being rod bipolar cells. By the end of the fourth postnatal week, the rod bipolar cells appeared mature, resembling those found in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal polypeptide modulates GABAA receptor function through activation of cyclic AMP

Vasoactive intestinal polypeptide (VIP) has been shown to potentiate current responses elicited by activation of the GABAA receptor (IGABA) in freshly dissociated ganglion cells of the rat retina. Here we tested the hypothesis that this heteroreceptor cross talk is mediated by an intracellular cascade of events that includes the sequential activation of a stimulatory guanine nucleotide binding (Gs) protein and adenylate cyclase, the subsequent increase in levels of cyclic AMP and, finally, the action of the cyclic AMP-dependent protein kinase (PKA). Intracellular dialysis of freshly dissociated ganglion cells with GTP gamma s irreversibly potentiated IGABA, while GDP beta s either decreased or had no effect on IGABA. Additionally, GDP beta s blocked the potentiation of IGABA by VIP. Cholera toxin rendered VIP ineffective in potentiating IGABA, while pertussis toxin had no effect on the VIP-induced potentiation of IGABA. Extracellular application of either forskolin or 8-bromo-cyclic AMP potentiated IGABA, as did the introduction of cyclic AMP directly into the intracellular compartment through the recording pipet. Intracellular application of cyclic AMP-dependent protein kinase (PKA) potentiated IGABA, while a PKA inhibitor blocked the potentiating effect of VIP. These results lead us to conclude that activation of a cyclic AMP-dependent second-messenger system mediates the modulation of GABAA receptor function by VIP in retinal ganglion cells.

Differentiation of a stem cell line toward a neuronal phenotype

This study examined the morphology and the development of inward currents in the course of differentiation of a stem cell toward a neuronal phenotype. Using the P19 embryonal cell line, whole‐cell current profiles of P19 cells before, during and after retinoic acid‐induced differentiation were matched with their morphology as well as with the expression of neuron‐specific enolase‐like immunoreactivity. Prior to and during the initial 48 hr of retinoic acid treatment, P19 cells either lacked detectable currents or expressed a voltage‐dependent outward potassium current, did not display neuron‐like morphology and did not express neuron‐specific enolase‐like immunoreactivity. Upon completion of retinoic acid treatment, the current profile of fully differentiated P19 cells was hallmarked by a large voltage‐dependent inward current which consisted of a sodium current and a smaller cobalt‐sensitive calcium component, in addition to the potassium current observed earlier. Such cells invariably emitted neuntes and displayed neuron‐specific enolase‐like immunoreactivity. Interestingly, coupling was prevalent among P19 cells in the undifferentiated state but was absent in the fully differentiated cultures. In studying cells undergoing neuronal differentiation, these results underscore the importance of taking into account both electrical properties and morphological considerations in determining the degree of differentiation.

A system of corticotropin releasing factor-containing amacrine cells in the rat retina

Immunohistochemical processing of Long-Evans retina wholemounts using an antiserum directed against rat, human corticotropin releasing factor revealed a group of immunoreactive amacrine cells. Two subpopulations could be distinguished based primarily on the location of their cell bodies. One subpopulation had cell bodies situated along the junction of the inner nuclear layer and the inner plexiform layer. The other subpopulation had cell bodies in the ganglion cell layer. The latter was judged to be displaced amacrine cells since double-label experiments indicated that the pattern of corticotropin releasing factor-like immunoreactive staining in the ganglion cell layer did not coincide with that of ganglion cells labeled retrogradely with fluorogold. Corticotropin releasing factor-like immunoreactive amacrine cells on either side of the inner plexiform layer emitted processes which ramified extensively in sublamina 5 and, to a lesser degree, in sublamina 4. A minority of these cells also sent a single process to ramify in sublamina 1. Throughout the retina, corticotropin releasing factor-like immunoreactive cells were distributed relatively evenly, with a tendency to peak in the superior temporal region. Despite the anatomical classification into two subpopulations, it is proposed that the corticotropin releasing factor-like immunoreactive cells are functionally one system, influencing preferentially synaptic interactions associated with the inner half of the inner plexiform layer. The results of this study provide anatomical basis for further investigations of corticotropin releasing factor as a putative peptidergic neurotransmitter in the retina.

Intraocular transplantation of cell layers derived from neonatal rat retina.

The goal of this study was to determine whether cell layers derived from either the inner or outer regions of the neonatal rat retina had the capacity to grow and differentiate when transplanted into the adult retina of the same species and, if so, whether there would be differences between the grafts originated from the two different cell populations. Two different tracers were used to label donor cells and to identify them following transplantation. Firstly, at postnatal day (PND) 2, the pups received bilateral injections of rhodamine-labeled microspheres in the superior colliculus in order to label retrogradely the retinal ganglion cells. From the day of birth until the day of sacrifice (PND 4), the donors received daily injections of [3H]thymidine to label the nuclei of dividing cells. At PND 4 the animals were sacrificed and the retinas isolated. Following brief enzymatic treatment, the inner and outer retinal regions were separated from each other using filter membranes. Cells derived from each of the two moieties were transplanted separately into the eyes of adult host animals of the same strain. After survival times ranging from 3 to 44 days, the host eyes were enucleated and prepared for examination using both light and electron microscopic methods. Both retinal regions gave viable transplants. Unexpectedly, the transplants derived from the inner zone of the retina survived better than those derived from the outer zone, despite the fact that the highest number of undifferentiated and mitotically active cells occurred in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

A vasoactive intestinal polypeptide system in retinal cell cultures: immunocytochemistry and physiology

The purpose of this study was to search for vasoactive intestinal polypeptide (VIP)-like immunoreactivity and VIP-mediated effects in cultures containing cells from the mammalian retina. VIP-like immunoreactivity was detected by indirect immunocytochemistry within 6 days after plating dissociated retinal cells from embryonic day-19 rats. In electrophysiological experiments, VIP was found to facilitate evoked transmission at cholinergic synapses formed by retinal neurons in culture.

Synapse-competence of LA-N-2 human neuroblastoma cells in coculture with rat striated muscle cells

The purpose of this study was to determine whether cells of the human neuroblastoma line, LA-N-2, are capable of establishing functional synapses in culture. We used a coculture system in which striated muscle cells from the rat served as postsynaptic targets for the cholinergic LA-N-2 cells. By recording postsynaptic responses from muscle cells, differentiated LA-N-2 cells were found to innervate muscle cells, releasing acetylcholine spontaneously at LA-N-2-muscle synapses. A subpopulation of the LA-N-2 cells forming synapses with the muscle cells also developed the ability to release acetylcholine in response to stimulation. This, coupled with results obtained from experiments examining the time course of synapse formation, led us to propose that the extent to which LA-N-2 cells in our coculture system are differentiated may vary and that this variation may underlie the degree to which they express neuron-like transmission properties.

Corticotropin releasing factor-like immunoreactivity (CRF-LI) in horizontal cells of the developing rat retina.

This study describes a phenomenon of transient expression of corticotropin releasing factor-like immunoreactivity (CRF-LI) in immature horizontal cells of the developing rat retina. These cells could be distinguished from those destined to become CRF-LI amacrine cells in the adult by their location within the outer portion of the neuroblastic layer (NBL) and by their ontogenetic pattern. Upon initial detection on postnatal day 3 (PD-3), faint CRF-LI cellular profiles were found in the outer portion of the NBL, limited to the central region of the retina. Subsequently, on PD-5, these profiles began to appear in the periphery, forming a single horizontal row along the outermost aspect of the developing inner nuclear layer (INL), concomitant with the establishment of the outer plexiform layer (OPL). The results of our birth-dating study combining immunohistochemistry and [3H]-thymidine autoradiography indicated that these cells were generated between embryonic day 14 and 18. These findings are consistent with them being horizontal cells. Between PD-7 and PD-9, CRF-LI in horizontal cells began to diminish progressively following a center-to-periphery gradient such that only sporadic, faintly immunoreactive patches of cells could be seen by the time of eye opening (PD-15). Around PD-19, it declined to levels below immunohistochemical detection. However, when rats were reared in complete darkness beginning at birth until PD-21, the period of CRF-LI expression in horizontal cells was prolonged and persisted throughout the first three postnatal weeks of development.

Avian retinal cells express enkephalin-like immunoreactivity in culture

Abstract A population of retinal cells derived from the embryonic chick acquire Leu-enkephalin-like immunoreactivity as they develop in cell culture.