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

This study was designed to characterize the influence of norepinephrine (NE), a putative cerebellar neurotransmitter, on immature synaptic activity during early postnatal periods of the developing cerebellar cortex. Norepinephrine was applied by microiontophoresis to single, young Purkinje cells to assess its effects on spontaneous and synaptically-mediated activities. Data from these studies indicate that the catecholamine exerts a profound modulatory-type influence on Purkinje cell responsiveness to activation of newly-established inputs. NE augmented climbing fiber-elicited excitatory burst responses by postnatal day 5 and enhanced ‘off-beam’ inhibitory activity by postnatal day 9, as soon as basket and stellate interneurons became functional. The functional implications of this noradrenergic facilitating effect, which is present throughout the maturation of the cerebellar cortical network, are discussed in terms of its possible contribution to the wiring of a definitive neuronal circuit.

The purpose of this study was to investigate the effect of γ-aminobutyric acid (GABA) on the function of synapses formed by cholinergic neurons derived from the chick retina. We used an experimental culture system in which striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This cell culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. By plating a low of density dissociated retinal cells with myotubes, it was possible to study relatively isolated, presynaptic cholinergic neurons. We found that GABA and agonists, muscimol and isoguvacine, inhibited spontaneous transmission at retina-muscle synapses. These inhibitory effects were reversibly blocked by bicuculline, a GABA receptor antagonist. The benzodiazepine, flurazepam, potentiated GABA-mediated inhibition. Overall, our findings suggest a direct inhibitory action of GABA on the cholinergic retinal neurons studied in our cell culture system.

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.

The levels of cAMP-dependent protein kinase (type I), or cGMP-dependent protein kinase, or protein I, and of a 23,000 MW substrate for the cGMP-dependent protein kinase were measured in cerebella from normal rats and in the cerebella from rats in which a selective loss of interneurons in the cerebellar cortex had been produced by X-irradiation. A decrease was observed in the concentrations of cAMP-dependent protein kinase and of protein I, whereas an increase was observed in the concentrations of cGMP-dependent protein kinase and of the 23,000 MW substrate. The data, taken together with the results of other studies, support the interpretation that cAMP-dependent protein kinase and protein I are distributed throughout the cerebellum, but that cGMP-dependent protein kinase and the 23,000 MW substrate are highly concentrated in Purkinje cells.

In this study, we examined the action of norepinephrine (NE) on developing cerebellar Purkinje cells. Responses of immature Purkinje cells to microiontophoretically applied NE were assessed both in terms of the direct depressant effect of the catecholamine as well as its ability to interact synergistically with γ-aminobutyric acid (GABA). Sensitivity for NE, GABA, taurine and β-alanine was found to be present by postnatal day 3. In addition, NE enhanced inhibitory responses of Purkinje cells to GABA but not those to β-alanine by at least postnatal day 5, before periods of extensive morphological differentiation and synaptic investment. The modulation by NE persisted into adulthood with no qualitative changes in characteristics. The results of this study support and extend the hypothesis that ‘chemosensitivity’ antedates synaptogenesis' to include responsiveness of developing Purkinje cells to interactions between putative cerebellar neurotransmitters.

The horseradish peroxidase neuronal staining technique was applied to reveal through anterograde and retrograde staining the detailed morphology of fiber arborization patterns in the cerebella of normal adult rats as well as those treated with neonatal X-irradiation. Morphological features of anterogradely-filled mossy fibers including the axonal arborization and glomerular-type synaptic specializations were found to be similar in all groups studied. In addition, retrogradely-filled Purkinje cell axon collaterals in the cerebella degranulated by X-irradiation exhibited much more elaborate branching and arborization of recurrent collaterals than those labeled in the normal cerebellar cortex. The presence of glomerular-type specializations in the degranulated cerebellum suggests that mossy fibers develop and maintain their normal features independent of major influences from granule cells. The hypertrophy observed after destruction of the cerebellar interneurons suggests the hypothesis that the growth of the normal Purkinje cell collateral system is normally suppressed by extrinsic factors within the neuronal circuit.

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.

The objective of this study was to investigate how the maturation of neurotransmission is regulated. We used a retina-muscle cell culture system to explore the effects of cyclic AMP analogues on the developmental step in which a presynaptic neuron acquires the ability to transmit excitatory information across a synapse. Cholinergic neurons dissociated from the perinatal rat retina form synapses in culture with rat striated muscle cells. Early in the functional maturation of these retina-muscle synapses, there is a period in which the release of acetylcholine occurs spontaneously, but cannot be evoked. This stage is followed by the emergence of transmitter release that is stimulus-evoked. We report here that exposure of cultured embryonic neurons of the rat retina to 8-bromo-cyclic AMP precociously induced in these neurons the ability to release acetylcholine at synapses in response to excitatory stimulation. This effect on synaptic development could be mimicked by an inhibitor of phosphodiesterase, isobutylmethylxanthine. The results of a variety of experiments lead us to propose that 8-bromo-cyclic AMP may accelerate the development of neurotransmission by influencing presynaptic events linking neuronal depolarization with acetylcholine release. Our data support the hypothesis that cyclic AMP may be an intracellular mediator for developmental signals which regulate the emergence of effective neurotransmission across nascent synapses.

The purpose of this study was to investigate the effect of dopamine on the function of synapses formed by cholinergic neurons derived from the rat retina. We used an experimental culture system in which rat striated muscle cells served as postsynaptic targets for cholinergic neurons of the retina. This culture system permitted the physiological monitoring of acetylcholine release at synapses formed by retinal neurons. We found that dopamine could facilitate evoked transmission at retina-muscle synapses. This facilitation by dopamine was reversible and could be blocked by haloperidol, a dopamine receptor antagonist. The adenosine 3':5'-phosphate analogue, 8-bromoadenosine 3':5'-phosphate, mimicked the facilitating effect of dopamine. In addition, dopamine elevated markedly the levels of adenosine 3':5'-phosphate in cultures of rat retinal cells. The results suggest that dopamine can regulate transmission through retinal neurons. Our findings support the hypothesis that a dopamine-induced facilitation of stimulus-evoked transmission involves the activation of dopamine receptors and the intracellular accumulation of adenosine 3':5'-phosphate.

Although beta 2-adrenergic receptors predominate in adult rat cerebellum, a small number of beta 1-receptors are also present. To investigate the cellular localization of beta 1- and beta 2-adrenergic receptors, the effects of intermittent neonatal X-irradiation focused on the cerebellum were determined on the densities of the two subtypes of beta-adrenergic receptor. This treatment destroys the late-maturing cerebellar interneurons including the granule, basket and stellate cells. A 60--70% loss of cerebellar mass was observed in 2-, 6- and 12-week-old animals. The large early-maturing Purkinje cells are resistant to the effects of X-irradiation. The total number of beta-adrenergic receptors per cerebellum, measured by Scatchard analysis of saturation isotherms of specific [125I]iodohydroxybenzylpindolol (IHYP) binding, was reduced by 70--80% in 2-, 6- and 12-week-old animals. The relative proportion of beta 1- and beta 2-receptors was determined by analyzing the inhibition of specific IHYP binding by the beta 1 selective antagonist practolol. The total number of beta 2-adrenergic receptors per cerebellum was reduced by 81--83% in 6- and 12-week-old X-irradiated rats. However, the number of beta 1-adrenergic receptors per cerebellum in 6- and 12-week-old X-irradiated rats was not significantly different from that in control animals. The results suggest that beta 2 receptors in the rat cerebellum are primarily associated with the small interneurons destroyed by neonatal X-irradiation. The beta 1 receptors may be located on a cell population which is unaffected by this treatment, possibly on cerebellar Purkinje 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.

Using patch-clamp recording in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we show in individual bipolar cells acutely dissociated from the adult rat retina a correlation between the expression of the GABAA receptor ρ1 subunit mRNA and a bicuculline-resistant, diazepam-insensitive component of the GABA-activated whole-cell current response. This “GABAC-like” response, contributing to approximately 42% of the GABA-activated whole-cell current and displaying variable sensitivity to picrotoxin, was found in bipolar cells but not in any of the ganglion cells examined. Expression profiling of GABAA receptor subunit mRNAs in individual electrophysiologically tested retinal neurons revealed that, while both bipolar cells and ganglion cells may express numerous GABAA receptor subunit isoforms, including that of ρ2, the expression of the ρ1 subunit was strictly limited to bipolar cells. We propose a possible link between the presence of a receptor with GABAC-like pharmacological profile and the expression of the retina-specific ρ1 subunit isoform. The results presented in this study constitute the first direct demonstration of such a correlation at the single-cell level.