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In the mature nervous system, changes in synaptic strength correlate with changes in neuronal structure. Members of the Nogo-66 receptor family have been implicated in regulating neuronal morphology. Nogo-66 receptor 1 (NgR1) supports binding of the myelin inhibitors Nogo-A, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), and is important for growth cone collapse in response to acutely presented inhibitorsin vitro. After injury to the corticospinal tract,NgR1limits axon collateral sprouting but is not important for blocking long-distance regenerative growthin vivo. Here, we report on a novel interaction between NgR1 and select members of the fibroblast growth factor (FGF) family. FGF1 and FGF2 bind directly and with high affinity to NgR1 but not to NgR2 or NgR3. In primary cortical neurons, ectopic NgR1 inhibits FGF2-elicited axonal branching. Loss ofNgR1results in altered spine morphologies along apical dendrites of hippocampal CA1 neuronsin vivo. Analysis of synaptosomal fractions revealed that NgR1 is enriched synaptically in the hippocampus. Physiological studies at Schaffer collateral–CA1 synapses uncovered a synaptic function for NgR1. Loss ofNgR1leads to FGF2-dependent enhancement of long-term potentiation (LTP) without altering basal synaptic transmission or short-term plasticity. NgR1 and FGF receptor 1 (FGFR1) are colocalized to synapses, and mechanistic studies revealed that FGFR kinase activity is necessary for FGF2-elicited enhancement of hippocampal LTP inNgR1mutants. In addition, loss ofNgR1attenuates long-term depression of synaptic transmission at Schaffer collateral–CA1 synapses. Together, our findings establish that physiological NgR1 signaling regulates activity-dependent synaptic strength and uncover neuronal NgR1 as a regulator of synaptic plasticity.

Vasoactive intestinal polypeptide (VIP) has been shown to potentiate current responses elicited by activation of the GABAAreceptor (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 GTPγsirreversibly potentiatedIGABA, while GDPßseither decreased or had no effect onIGABA. Additionally, GDPßsblocked the potentiation ofIGABAby VIP. Cholera toxin rendered VIP ineffective in potentiatingIGABA, while pertussis toxin had no effect on the VIP-induced potentiation ofIGABA. Extracellular application of either forskolin or 8-bromo-cyclic AMP potentiatedIGABA, 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) potentiatedIGABA, 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 GABAAreceptor function by VIP in retinal ganglion cells.

This study profiled the expression of the family of GABAA receptor β-subunits in the adult rat retina. Using a combination of reverse transcriptase reaction followed by polymerase chain reaction (RT-PCR) with gene-specific primers, the expression of mRNAs encoding the β1, β2, and β3 subunits was first examined in the intact retina and then in separated retinal nuclear layers. However, it was found that a critical analysis. had to be carried out at the level of the single cell in order to resolve the differential patterns of expression among the retinal cell types. When cells were isolated and identified following acute dissociation, RT-PCR revealed that individual rod photoreceptor cells expressed consistently the β1 and β2 messages while the bipolar cells expressed the β1 and β3 messages. Ganglion cells displayed considerable variability in β-subunit expression, perhaps reflecting their functional and morphological heterogeneity in the retina. In contrast, the nonneuronal Mueller cells did not express any of the β-subunit messages. These results indicate that the expression of GABAA receptor subunits is cell-type dependent. Furthermore, as the expression of other families of GABAA receptor subunits are profiled and the patterns of subunit assembly are better understood, our results raise the possibility that GABAA receptors with different subunit compositions can be expected to be coexpressed within a single retinal neuron.

We present here a method for broadly characterizing single cells at the molecular level beyond the more common morphological and transmitter/receptor classifications. The RNA from defined single cells is amplified by microinjecting primer, nucleotides, and enzyme into acutely dissociated cells from a defined region of rat brain. Further processing yields amplified antisense RNA. A second round of amplification results in greater than 10(6)-fold amplification of the original starting material, which is adequate for analysis--e.g., use as a probe, making of cDNA libraries, etc. We demonstrate this method by constructing expression profiles of single live cells from rat hippocampus. This profiling suggests that cells that appear to be morphologically similar may show marked differences in patterns of expression. In addition, we characterize several mRNAs from a single cell, some of which were previously undescribed, perhaps due to "rarity" when averaged over many cell types. Electrophysiological analysis coupled with molecular biology within the same cell will facilitate a better understanding of how changes at the molecular level are manifested in functional properties. This approach should be applicable to a wide variety of studies, including development, mutant models, aging, and neurodegenerative disease.

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 recieved 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 [ 3 H]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 adn 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. The results indicate that the ‘sandwich’ method of layer separation offers a fast, convenient, and reliable means of obtaining enriched cell populations from the perinatal mammalian retina for purposes of grafting and studying their growth.

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)

This paper describes experiments on GABA-activated whole-cell membrane currents in bipolar cells freshly isolated from the adult rat retina. The main goal was to determine whether bipolar cell responses to GABA could be resolved in terms of mediation by the GABAA receptor, the GABAB receptor, or both. Bipolar cells were isolated by gentle enzymatic dissociation and identified by their distinct morphology. GABA agonists and antagonists were applied focally by pressure and the resultant currents were recorded under whole-cell voltage clamp. In all bipolar cells tested, GABA (0.1–100 μM) induced a monophasic response associated with a conductance increase (IGABA). The shift in reversal potential for IGABA as a function of pipet [CI] paralleled that predicted based on the Nernst equation for Cl−. IGABA was mimicked by muscimol (5–20 μM) and antagonized by bicuculline (20–100 μM). Baclofen (0.1–1.0 mM) produced no apparent conductance change. “Hot spots” of sensitivity to GABA which might be associated with regions of synaptic contact were not found; both the soma and processes of all bipolar cells were responsive to focally applied GABA. Furthermore, all bipolar cells tested responded to glycine.In conclusion, we have established the presence of GABAA receptors on rat retinal bipolar cells. Our data suggest further that these cells lack GABAB receptors. Finally, our observation that bipolar cells in the rat retina are relatively homogeneous in terms of their sensitivity to GABA and glycine lead us to postulate that the functional significance of the presence of receptors and their distribution on a neuron may be dictated more by the topography of the presynaptic inputs than by its inherent chemosensitivity.

This study examined the effect of chronic exposure to ethanol on gamma-aminobutyric acid type-A (GABA(A)) receptor-mediated responses of layer II pyramidal neurons of the piriform cortex. Slices containing the piriform cortex were derived from pair-fed adult rats maintained on ethanol-supplemented or control liquid diet for 30 days. Responses of identified layer II pyramidal neurons to exogenously applied GABA were monitored by whole-cell patch-clamp recording. Chronic exposure to ethanol resulted in a rightward shift in the EC(50) of GABA and a decrease in the amplitude of maximal GABA response. GABA-induced responses were modulated by acutely applied ethanol (10-100 mM) in both chronic ethanol-treated and control groups. No significant difference was found in the average change in GABA response, suggesting that tolerance to acute ethanol exposure did not develop. When the modulatory responses of individual cells were classified and grouped as either being attenuating, potentiating, or having no effect, the incidence of potentiation in the ethanol-treated group was significantly higher. Consistent with the absence of tolerance to acute ethanol, cross-tolerance to diazepam was not observed following 30 days of treatment with ethanol. These results are discussed in light of regionally specific effects of chronic ethanol treatment on GABA(A) receptor-mediated responses of layer II piriform cortical neurons.

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell developmentin vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABAAreceptor β2 subunit. This increase in subunit expression is paralleled by an increase in functional GABAAreceptors. In contrast to its effects on GABAAreceptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABAAreceptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processesin vivo.

This study examined the effect of ethanol on responses of primary somatosensory cortical neurons to AMPA. Thin (200-250 microns) brain slices were sectioned to include the primary somatosensory cortex of rats 6-15 days after birth. Visually-identified neurons were selected for whole-cell patch clamp recording and an eight-barrel drug pipet assembly was used to deliver test agents. Ethanol (5-100 mM) either positively or negatively modulated AMPA (100 microM)-induced current to varying degrees in approximately 70% of primary somatosensory cortical neurons. As revealed in layer V large pyramidal neurons, the outcome of an ethanol-induced modulation appeared to be age-dependent, the trend being one of potentiation in slices derived from younger rats (postnatal days 6-9) but one of attenuation in those derived from older animals (postnatal days 13-15). These findings indicate that ethanol at physiologically relevant concentrations modulates non-NMDA receptor-mediated responses of neurons in the rat primary somatosensory cortex.

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m 1 and m 2 but not for the m 3 to m 5 mAChRs. The identity of the m 1 and m 2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m 1 and m 2 , but not m 3 , mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m 1 mAChR was responsible for the stimulatory effects on Ca 2+ transients by high concentrations of carbachol (>10 μmol/L) known to occur in these cells. In pertussis toxin–treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 μmol/L) increased the basal Ca 2+ level from 96±7 to 118±8 nmol/L and the peak Ca 2+ transient level from 519±32 to 640±36 nmol/L (mean±SEM, P 2+ transients were antagonized by 10 nmol/L pirenzepine, an m 1 mAChR–selective antagonist. In contrast, the m 2 mAChR–selective antagonist methoctramine (up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m 1 mAChRs are expressed on adult rat ventricular myocytes in addition to m 2 mAChRs. The results further suggest that m 1 mAChRs mediate the stimulatory responses on Ca 2+ transients to high concentrations of cholinergic agonists seen in these cells.

Substance-P-like immunoreactivity (SP-LI) cells in the Long-Evans rat retina were investigated by combining immunohistochemistry with [3H]thymidine autoradiography. Two subpopulations of SP-LI amacrine cells, with cell bodies in either the proximal portion of the inner nuclear layer (INL) or the ganglion cell layer (GCL), were identified based on morphology, pattern of distribution and development. In the INL, SP-LI cells were found scattered throughout the retina. However, in the GCL, they were limited to the superio-temporal region. Such a contrast in distribution specific to nuclear layers was present upon first detection of SP-LI amacrine cells and persisted throughout development. Birthdating revealed a temporal lag in the histogenesis of SP-LI cells situated in the GCL relative to that in the INL, suggesting that the two subpopulations developed separately. Overall, unique anatomical features of the SP-LI amacrine cells in the rat retina were observed which could only have been uncovered through detailed analyses in the adult as well as during postnatal development.

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 theinner 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. Overall, more dynamic changes occurred at the axon terminal bulbs than at the cell bodies during the maturational process of rod bipolar cells. Interestingly, PKIC-LI was expressed precociusly in these cells when rat pups were reared in complete darkness starting from the day of birth.

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.

The histogenesis of amacrine cells expressing corticotropin releasing factor-like immunoreactivity (CRF-LI) was examined in the rat retina by incorporating [ 3 H]thymidine autoradiography and immunohistochemistry. Our results indicate that, during the period of amacrine cell generation, the population of CRF-LI amacrine cells are generated within a window of time from gestational days (ED)-16 to -20; the majority of them are produced by ED-18. No CRF-LI cells have been found to be ‘born’ postnatally. The histogenetic pattern of these cells follows a center-to-periphery gradient. Furthermore, CRF-LI cells in both the inner nuclear layer and the ganglion cell layer are generated in the same histogenetic wave and follow an identical temporo-spatial pattern. These results are consistent with and extend our previous findings of a differential change in CRF-LI cell density and total cell number during postnatal development.

The postnatal development of the corticotropin releasing factor-like immunoreactive (CRF-LI) amacrine cells was investigated in the Long—Evans rat retina. The pattern of development of CRF-LI cells was studied by immunohistochemistry, their cell number and density throughout the first two weeks of postnatal development were analyzed, and correlative measures of CRF-LI content were obtained using radioimmunoassay (RIA). The overall pattern of CRF-LI development, as revealed by either method, is characterized initially by faint staining and low content, respectively, which began to increase in staining intensity and content until a peak was reached around postnatal day (PD)-15, the time of eye opening. In determining cell number and density, emphasis was placed on the relationship between the development of CRF-LI neurons in the inner nuclear layer (INL) and that in the ganglion cell layer (GCL). Such quantitative analyses revealed a series of dynamic shifts in the distribution of CRF-LI cell density in both a horizontal orientation and a vertical orientation prior to PD-15. Horizontally, the shift involved a center-to-periphery density gradient which disappeared progressively as the retina matured. Vertically, a reciprocal change in total cell number occurred; the number of CRF-LI cells in the INL decreased while that in the GCL increased. These changes stabilized by PD-15 and, by PD-19, the CRF-LI cells appeared morphologically mature.

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 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.