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Fetal alcohol spectrum disorder (FASD) encompasses a range of cognitive and behavioral deficits, with aberrances in the function of cerebral cortical pyramidal neurons implicated in its pathology. However, the mechanisms underlying these aberrances, including whether they persist well beyond ethanol exposure in utero, remain to be explored. We addressed these issues by employing a mouse model of FASD in which pregnant mice were exposed to binge-type ethanol from embryonic day 13.5 through 16.5. In both male and female offspring (postnatal day 28–32), whole-cell patch clamp recording of layer V/VI somatosensory cortex pyramidal neurons revealed increases in the frequency of excitatory and inhibitory postsynaptic currents. Furthermore, expressing channelrhodopsin in either GABAergic interneurons (Nkx2.1Cre-Ai32) or glutamatergic pyramidal neurons (Emx1IRES Cre-Ai32) revealed a shift in optically evoked paired-pulse ratio. These findings are consistent with an excitatory-inhibitory imbalance with prenatal ethanol exposure due to diminished inhibitory but enhanced excitatory synaptic strength. Prenatal ethanol exposure also altered the density and morphology of spines along the apical dendrites of pyramidal neurons. Thus, while both presynaptic and postsynaptic mechanisms are affected following prenatal exposure to ethanol, there is a prominent presynaptic component that contributes to altered inhibitory and excitatory synaptic transmission in the somatosensory cortex.

Exposure of the fetus to alcohol (ethanol) via maternal consumption during pregnancy can result in fetal alcohol spectrum disorders (FASD), hallmarked by long-term physical, behavioral, and intellectual abnormalities. In our preclinical mouse model of FASD, prenatal ethanol exposure disrupts tangential migration of corticopetal GABAergic interneurons (GINs) in the embryonic medial prefrontal cortex (mPFC). We postulated that ethanol perturbed the normal pattern of tangential migration via enhancing GABAAreceptor-mediated membrane depolarization that prevails during embryonic development in GABAergic cortical interneurons. However, beyond this, our understanding of the underlying mechanisms is incomplete. Here, we tested the hypothesis that the ethanol-enhanced depolarization triggers downstream an increase in high-voltage-activated nifedipine-sensitive L-type calcium channel (LTCC) activity and provide evidence implicating calcium dynamics in the signaling scheme underlying the migration of embryonic GINs and its aberrance. Tangentially migrating Nkx2.1+GINs expressed immunoreactivity to Cav1.2, the canonical neuronal isoform of the L-type calcium channel. Prenatal ethanol exposure did not alter its protein expression profile in the embryonic mPFC. However, exposing ethanol concomitantly with the LTCC blocker nifedipine prevented the ethanol-induced aberrant migration bothin vitroandin vivo. In addition, whole-cell patch clamp recording of LTCCs in GINs migrating in embryonic mPFC slices revealed that acutely applied ethanol potentiated LTCC activity in migrating GINs. Based on evidence reported in the present study, we conclude that calcium is an important intracellular intermediary downstream of GABAAreceptor-mediated depolarization in the mechanistic scheme of an ethanol-induced aberrant tangential migration of embryonic GABAergic cortical interneurons.

Prenatal exposure to ethanol induces aberrant tangential migration of corticopetal GABAergic interneurons, and long-term alterations in the form and function of the prefrontal cortex. We have hypothesized that interneuronopathy contributes significantly to the pathoetiology of fetal alcohol spectrum disorders (FASD). Activity-dependent tangential migration of GABAergic cortical neurons is driven by depolarizing responses to ambient GABA present in the cortical enclave. We found that ethanol exposure potentiates the depolarizing action of GABA in GABAergic cortical interneurons of the embryonic mouse brain. Pharmacological antagonism of the cotransporter NKCC1 mitigated ethanol-induced potentiation of GABA depolarization and prevented aberrant patterns of tangential migration induced by ethanol in vitro. In a model of FASD, maternal bumetanide treatment prevented interneuronopathy in the prefrontal cortex of ethanol exposed offspring, including deficits in behavioral flexibility. These findings position interneuronopathy as a mechanism of FASD symptomatology, and posit NKCC1 as a pharmacological target for the management of FASD.

Epidemiological studies have reported a reduction in the prevalence of Alzheimer's disease in individuals that ingest low amounts of alcohol. Also, it has been found that moderate consumption of ethanol might protect against β-amyloid (Aβ) toxicity. However, the mechanism underlying its potential neuroprotection is largely unknown. In the present study, we found that ethanol improved the cognitive processes of learning and memory in 3xTgAD mice. In addition, we found that a low concentration of ethanol (equivalent to moderate ethanol consumption) decreased the binding of Aβ (1 and 5 μM) to neuronal membranes and, consequently, its synaptotoxic effect in rat hippocampal and cortical neurons under acute (30 minutes) and chronic (24 hours) incubation conditions. This effect appears to be exerted by a direct action of ethanol on Aβ because electron microscopy studies showed that ethanol altered the degree of Aβ aggregation. The action of ethanol on Aβ also prevented the peptide from perforating the neuronal membrane, as assayed with patch clamp experiments. Taken together, these results contribute to elucidating the mechanism by which low concentrations of ethanol protect against toxicity induced by Aβ oligomers in primary neuronal cultures. These results may also provide an explanation for the decrease in the risk of Alzheimer's disease in people who consume moderate doses of alcohol.

Neurotrophins have been implicated in regulating neuronal differentiation, promoting neuronal survival, and modulating synaptic efficacy and plasticity. The prevailing view is that, depending on the target and mode of action, most neurotrophins can be trafficked and released either anterogradely or retrogradely in an activity-dependent manner. However, the prototypic neurotrophin, nerve growth factor (NGF), is not thought to be anterogradely delivered. Here we provide the neuroanatomical substrate for an anterograde hippocamposeptal transport of NGF by demonstrating its presence in mouse hippocampal GABAergic neurons and in their hippocamposeptal axons that ramify densely and abut neurons in the medial septum/diagonal band of Broca (MS/DB). We also demonstrate an activity-dependent increase in septal NGF levels that is dependent on the pattern of intrahippocampal stimulation. In addition, we show that acute exposure to NGF, via activation of TrkA, attenuates GABAAreceptor-mediated inhibitory synaptic currents and reduces sensitivity to exogenously applied GABA. These acute actions of NGF display cell type and functional selectivity insofar as (1) they were found in cholinergic, but not GABAergic, MS/DB neurons, and (2) glutamate-mediated excitatory synaptic activity as well as AMPA-activated current responses were unaffected. Our results advocate a novel anterograde, TrkA-mediated NGF signaling in the CNS.

During rodent corticogenesis, a sizeable subpopulation of γ-aminobutyric acid (GABA)ergic interneurons arises extracortically from the medial ganglionic eminence (MGE). These neurons progressively acquire responsiveness to GABA in the course of corticopetal tangential migration, a process regulated by ambient GABA and mediated by GABA(A) receptors. Here, we combined patch clamp electrophysiology and single-cell reverse transcription-polymerase chain reaction to examine GABA(A) receptor expression in green fluorescent MGE-derived (eGFP+) cells in telencephalic slices from gestational day 14.5 BAC-Lhx6 embryos. GABA concentration-response curves revealed lower apparent affinity and efficacy in eGFP+ cells in and around the MGE than their counterparts in the cortex. Pharmacological tests revealed subunit-selective response profiles in the MGE and cortex consistent with differential expression of GABA(A) receptor isoforms. Profiling of GABA(A) receptor subunit transcripts (α1-5, β1-3, and γ1-3, δ) uncovered increased expression of the α1-, α2-, α5-, γ2-, and γ3-subunit messenger RNAs in the cortex. We propose that the dynamic expression of certain GABA(A) receptor subunits contributes to assembling receptor isoforms that confer functional attributes important in regulating the migration and maturation of primordial GABAergic cortical interneurons.

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.

Background Exposure to ethanol (EtOH) in utero alters the disposition of tangentially migrating GABAergic interneurons in the fetal brain. The medial ganglionic eminence (MGE) gives rise to a large portion of cortical GABAergic interneurons, including the parvalbumin-expressing interneurons that shape and contribute to inhibitory/excitatory (I/E) balance of the intracortical circuit. Here, we investigated in the mouse medial prefrontal cortex (mPFC) the hypothesis that low levels of maternal EtOH consumption from closure of the neural tube embryonic day (E) 9.5 until birth result in an enduring interneuronopathy. Methods Pregnant mice were subjected to a 2% w/w EtOH consumption regimen starting at neural tube closure and ending at parturition. Neurogenesis in the MGE was assessed by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence at E12.5. The count and distribution of parvalbumin-expressing interneurons were determined in adult animals, and patch clamp electrophysiology was performed to determine GABAergic function and I/E balance. Open-field behavior in adult mice was assessed to determine whether the EtOH-exposed cohort displayed a lasting alteration in exploratory behavior. Results In embryos exposed to EtOH in utero, we found increased BrdU labeling in the MGE, pointing to increased neurogenesis. Adult mice prenatally exposed to EtOH were hyperactive, and this was associated with an increase in parvalbumin-expressing GABAergic interneurons in the mPFC. In addition, prenatal EtOH exposure altered the balance between spontaneous inhibitory and excitatory synaptic input and attenuated GABAergic tone in layer V mPFC pyramidal neurons in juvenile mice. Conclusions These findings underscore that altered migration of GABAergic interneurons contributes to the EtOH-induced aberration of cortical development and that these effects persist into adulthood as altered cortical form and function.

During corticogenesis, cells from the medial ganglionic eminence (MGE) migrate tangentially into the neocortical anlage. Here we report that gamma-aminobutyric acid (GABA), via GABAA receptors, regulates tangential migration. In embryonic telencephalic slices, bicuculline produced an outward current in migrating MGE-derived cells in the neocortex, suggesting the presence of and tonic activation by ambient GABA. Ambient GABA was also present in the MGE, although this required demonstration using as bioassay HEK293 cells expressing high-affinity alpha6/beta2/gamma2s recombinant GABAA receptors. The concentration of ambient GABA was 0.5+/-0.1 microM in both regions. MGE-derived cells before the corticostriate juncture (CSJ) were less responsive to GABA than those in the neocortex, and profiling of GABAA receptor subunit transcripts revealed different expression patterns in the MGE vis-à-vis the neocortex. These findings suggest a dynamic expression of GABAA receptor number or isoform as MGE-derived cells enter the neocortex and become tonically influenced by ambient GABA. Treatment with bicuculline or antibody against GABA did not affect migration of MGE-derived cells before the CSJ but decreased "crossing index," reflecting impeded migration past the CSJ into the neocortex. Treatment with diazepam or addition of exogenous GABA increased crossing index. We conclude that ambient GABA promotes cortical entry of tangentially migrating MGE-derived cells.

Nerve growth factor (NGF) has been implicated in maintaining and regulating normal functioning of the septohippocampal pathway. However, many aspects of its physiological actions and the underlying mechanisms await elucidation. In this study, we investigated the effect of acute NGF exposure on neurons in the mouse medial septum/diagonal band of Broca (MS/DB), focusing on the cholinergic neurons and the subpopulation of noncholinergic neurons that were identified to be putatively GABAergic. We report that MS/DB neurons in a thin slice preparation, when exposed to NGF via bath perfusion, rapidly and indiscriminately increased the rate of spontaneous firing in all MS/DB neurons. However, focal application of NGF to individual MS/DB neurons increased spontaneous firing in cholinergic, but not in the noncholinergic, subpopulation. The NGF-induced effect on cholinergic neurons was direct, requiring activation and signaling via TrkA receptors, which were immunohistochemically localized to the cholinergic neurons in the MS/DB. TrkA receptors were absent in putative GABAergic MS/DB neurons, and blockade of TrkA signaling in these and other noncholinergic neurons had no effect on their firing activity after exposure to NGF. Conversely, methyl scopolamine, blocked the increased firing activity of noncholinergic neurons during bath perfusion of NGF. We propose a cell type-specific mode of action for NGF in the MS/DB. The neurotrophin directly enhances cholinergic neuronal activity in the MS/DB through TrkA-mediated signaling, increasing acetylcholine release and, thus, muscarinic tone. This increase in muscarinic tone, in turn, results in heightened firing activity in noncholinergic MS/DB neurons.

Brain-derived neurotrophic factor (BDNF) regulates neuronal survival, neurite outgrowth, and excitatory synaptic transmission. We reported recently that acute BDNF exposure decreased gamma-aminobutyric acid (GABA) responses in cultured mouse cerebellar granule cells through tyrosine receptor kinase B (TrkB) receptor-mediated signaling. In the present study, we extend this work to investigate BDNF-induced modulation of GABA responses and GABA(A) receptor-mediated synaptic events in cerebellar slices. Thin (200 microm) parasagittal slices of cerebellum were prepared from postnatal Day 7 and 14 mice. Purkinje cells and granule cells, both of which express TrkB-like immunoreactivity, were identified for whole-cell recording. BDNF promptly enhanced GABA responses in Purkinje cells but, consistent with our previous finding in culture, attenuated those recorded in granule cells. In Purkinje cells, BDNF exposure shifted rightward the cumulative peak amplitude distribution for miniature inhibitory postsynaptic currents (mIPSCs) without changing the mIPSC frequency. BDNF-induced potentiation of Purkinje cell responses to GABA was blocked by TrkB-Fc (receptor body that sequesters BDNF), K252a (inhibitor of TrkB receptor autophosphorylation), U73122 (inhibitor of phospholipase-Cgamma [PLCgamma]), KN62 (specific inhibitor of calcium/calmodulin-dependent kinase), KT5720 (specific cyclic AMP-dependent kinase inhibitor), and by intracellular dialysis of Rp-cyclic AMP or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid). Overall, our results indicate that BDNF acutely potentiates GABA(A) receptor function in cerebellar Purkinje cells via the TrkB receptor-PLCgamma signal transduction cascade. In addition, we propose that cyclic AMP-mediated intracellular signaling mechanisms may facilitate manifestation of the BDNF-induced modulatory outcome.

This study examined the effect of chronic exposure to ethanol and brain-derived neurotrophic factor (BDNF) on the responsiveness of cerebellar granule cells to γ-aminobutyric acid (GABA). Cerebellar granule cell cultures were chronically exposed to ethanol (100 mM), BDNF (20 ng/ml), or the combination of ethanol and BDNF. Whole-cell current responses of granule cells to exogenously applied GABA were monitored following at least 5 days of chronic exposure. In the ethanol-treated cultures, granule cell responsiveness to GABA was attenuated. Concomitant exposure of cultures to ethanol and BDNF mitigated the ethanol-induced attenuation of GABA response, although BDNF, by itself, did not affect responsiveness to GABA. BDNF increased the expression of the GABAA receptor α6 subunit, whereas ethanol had no effect, in chronically treated granule cell cultures. In addition, concomitant treatment with BDNF and ethanol did not increase the expression of the GABAA receptor α6 subunit, so the subunit expression alone could not account for the mitigating effect of BDNF. We propose that different mechanisms regulating responsiveness to GABA underlie the effects induced by ethanol and BDNF, with the former influencing the expression of functional GABAA receptors and the latter involving the activation of the TrkB receptor and its downstream signaling pathways. © 2003 Wiley-Liss, Inc.

In addition to exerting long-term neurotrophic influences on developmental process such as neuronal survival and neuritic outgrowth, brain-derived neurotrophic factor (BDNF) has been reported to modulate synaptic transmission in the short-term. Considerable evidence indicates that BDNF acutely modulates NMDA receptor-mediated synaptic activity. However, whether BDNF modulates inhibitory synaptic transmission remains to be firmly established. In the present study, we examined the effect of acute BDNF exposure on GABA-evoked whole-cell responses as well as GABAergic synaptic activity in cultured mouse cerebellar granule cells. GABA-evoked responses were reduced by 39.5 +/- 4.7 % upon acute and focal application of BDNF (100 ng ml-1). The reduction of the GABA response recovered only partially even minutes after removal of BDNF. TrkB-IgG and K252a, but not K252b, prevented the BDNF-induced attenuation of the GABA response. BDNF exposure shifted the cumulative peak amplitude distribution leftward for both spontaneous IPSCs (sIPSCs) and miniature IPSCs (mIPSCs) without affecting the rise time and decay time constants. Acute exposure to BDNF also resulted in internalization of GABAA receptors in cultured cerebellar granule cells, as reflected by diminished immunostaining with an antibody against the GABAA receptor beta2/3 subunit. Although the BDNF-induced GABAA receptor internalization was sensitive to K252a, it did not become manifest until 5 min after exposure to BDNF. Therefore, receptor internalization alone cannot account for the prompt BDNF-induced attenuation of GABA-mediated activity. We conclude that BDNF modulates GABAA receptor-mediated activity through TrkB receptor signalling that triggers a kinase-dependent short latency effect and a delayed longer latency effect hallmarked by receptor internalization.

Exposure to ethanol in utero may result in fetal alcohol spectrum disorder (FASD), hallmarked by varying degrees of cognitive deficits, many of which are associated with abnormalities in processing within the cortical circuitry (Sadrian et al., 2013; Shawa et al., 2013). Developmentally, the formation of cortical circuits is a tightly regulated sequel of events involving genesis, migration, and integration of inhibitory and excitatory neurons. Cajal–Retzius cells, as the transient and first-born neurons of the embryonic rodent cortex, arise from pallial and subpallial origins and migrate tangentially below the pial surface to cover the entire neocortex (Bielle et al., 2005; Garcia-Moreno et al., 2007). They release the glycoprotein reelin (D'Arcangelo et al., 1995; Hirotsune et al., 1995; Ogawa et al., 1995), implicated in regulating neuronal migration, layering, and neurogenesis in the developing cortex (Franco et al., 2011; Frotscher, 1998; Gil-Sanz et al., 2013; Gupta et al., 2003; Jossin and Cooper, 2011; Lakoma et al., 2011; Olson et al., 2006; Sekine et al., 2011). Thus, Cajal–Retzius cells are in a favorable position to orchestrate early aspects of corticogenesis, cortical patterning, and the establishment of nascent cortical circuits (Schwartz et al., 1998; Soriano and Del Rio, 2005). While the importance of Cajal–Retzius cells in corticogenesis is well-documented (Super et al., 2000), whether they are susceptible to ethanol exposure in utero is unexplored. In this study, pregnant mice were administered ethanol in a liquid diet that yielded moderate levels of blood alcohol (x = 20 mg/dl). The adolescent offspring exposed in utero to ethanol displayed altered open-field activity, indicating a relevant neurobehavioral consequence of our in utero ethanol exposure paradigm. We started the maternal consumption regimen on embryonic day (E)9, the time of closure of the neural tube, to span the entire period of cortical development. In this light, beyond regulating migration and neuronal integration during corticogenesis, Cajal–Retzius cells have also been postulated to play a functional role within the developing cortical circuit (Aguilo et al., 1999; Radnikow et al., 2002; Soda et al., 2003). We show for the first time that they receive GABAergic synaptic input as early as E13.5 in the mouse neocortex. In addition, we report that ethanol exposure in utero early on in cortical development alters the genesis, distribution, and GABAergic synaptic input of Cajal–Retzius cells.

This study examined glutamate-activated current responses of mouse and human Cajal-Retzius (C-R) cells. Thin cortical slices were prepared from the brains of mice 4–6 days after birth and from those of midgestational human fetuses. Both human and mouse C-R cells displayed glutamate-induced whole-cell current responses that were voltage-dependent and included an N-methyl-d-aspartate (NMDA) receptor–mediated component that was differentially sensitive to blockade by the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid and ifenprodil. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA glutamate receptor agonist, induced current responses in human but not in mouse C-R cells. These results, taken together, lead us to conclude that human C-R cells express both NMDA and AMPA types of glutamate receptors very early during development of the cortex. In contrast, mouse C-R cells express only the NMDA type of glutamate receptor. Thus we demonstrate a species-dependent sensitivity of C-R cells to glutamate and postulate that this differential sensitivity may account in part for a species-dependent difference in the persistence of C-R cells during cortical development.

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 septohippocampal system has been implicated in the cognitive deficits associated with ethanol consumption, but the cellular basis of ethanol action awaits full elucidation. In the medial septum/diagonal band of Broca (MS/DB), a muscarinic tone, reflective of firing activity of resident cholinergic neurons, regulates that of their noncholinergic, putatively GABAergic, counterparts. Here we tested the hypothesis that ethanol alters this muscarinic tone. The spontaneous firing activity of cholinergic and noncholinergic MS/DB neurons were monitored in acute MS/DB slices from C57Bl/6 mice. Exposing the entire slice to ethanol increased firing in both cholinergic and noncholinergic neurons. However, applying ethanol focally to individual MS/DB neurons increased firing only in cholinergic neurons. The differential outcome suggested different mechanisms of ethanol action on cholinergic and noncholinergic neurons. Indeed, with bath-perfused ethanol, the muscarinic antagonist methyl scopolamine prevented the increase in firing in noncholinergic, but not cholinergic, MS/DB neurons. Thus, the effect on noncholinergic neuronal firing was secondary to ethanol's direct action of acutely increasing muscarinic tone. We propose that the acute ethanol-induced elevation of muscarinic tone in the MS/DB contributes to the altered net flow of neuronal activity in the septohippocampal system that underlies compromised cognitive function.

ACh release into the rodent prefrontal cortex is predictive of successful performance of cue detection tasks, yet the cellular mechanisms underlying cholinergic modulation of cortical function are not fully understood. Prolonged (“tonic”) muscarinic ACh receptor (mAChR) activation increases the excitability of cortical pyramidal neurons, whereas transient (“phasic”) mAChR activation generates inhibitory and/or excitatory responses, depending on neuron subtype. These cholinergic effects result from activation of “M1-like” mAChRs (M1, M3, and M5 receptors), but the specific receptor subtypes involved are not known. We recorded from cortical pyramidal neurons from wild-type mice and mice lacking M1, M3, and/or M5 receptors to determine the relative contribution of M1-like mAChRs to cholinergic signaling in the mouse prefrontal cortex. Wild-type neurons in layer 5 were excited by tonic mAChR stimulation, and had biphasic inhibitory followed by excitatory, responses to phasic ACh application. Pyramidal neurons in layer 2/3 were substantially less responsive to tonic and phasic cholinergic input. Cholinergic effects were largely absent in neurons from mice lacking M1 receptors, but most were robust in neurons lacking M3, M5, or both M3 and M5 receptors. The exception was tonic cholinergic suppression of the afterhyperpolarization in layer 5 neurons, which was absent in cells lacking either M1 or M3 receptors. Finally, we confirm a role for M1 receptors in behavior by demonstrating cue detection deficits in M1-lacking mice. Together, our results demonstrate that M1 receptors facilitate cue detection behaviors and are both necessary and sufficient for most direct effects of ACh on pyramidal neuron excitability.

Understanding the biology of adult neural stem cells has important implications for nervous system development and may contribute to our understanding of neurodegenerative disorders and their treatment. We have characterized the process of olfactory neurogenesis in adult mice lacking inhibitor of DNA binding 2−/−(Id2−/−). We found a diminished olfactory bulb containing reduced numbers of granular and periglomerular neurons with a distinct paucity of dopaminergic periglomerular neurons. While no deficiency of the stem cell compartment was detectable, migrating neuroblasts inId2−/−mutant mice prematurely undergo astroglial differentiation within a disorganized rostral migratory stream. Further, when evaluatedin vitroloss ofId2results in decreased proliferation of neural progenitors and decreased expression of theHes1andAscl1 (Mash1)transcription factors, known mediators of neuronal differentiation. These data support a novel role for sustainedId2expression in migrating neural progenitors mediating olfactory dopaminergic neuronal differentiation in adult animals.

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.

Cajal-Retzius cells are implicated in regulating neuronal migration and lamination during corticogenesis. In rodents, Cajal-Retzius cells are transient, being prevalent in the marginal zone of the embryonic neocortex and declining over the first two postnatal weeks. While studies have examined in postnatal neocortex the properties of GABA(A) receptors in Cajal-Retzius cells, less is known about their disposition at embryonic stages. Here, we combined patch-clamp electrophysiology and single-cell mRNA profiling to probe the expression of GABA(A) receptors in Cajal-Retzius cells. In embryonic neocortical slices, GABA elicited GABA(A) receptor-mediated current responses that were diazepam-insensitive and inhibited by Zn(2+), a pharmacological profile consistent with expression of gamma-less GABA(A) receptor isoforms. Non-Cajal-Retzius cells in the same embryonic slices, on the other hand, were robustly potentiated by diazepam and were insensitive to Zn(2+), typical of gamma-containing GABA(A) receptor isoforms, as were Cajal-Retzius cells in the postnatal neocortex. Single-cell mRNA profiling and immunohistochemistry confirmed expression of GABA(A) receptor gamma subunit transcript and protein, respectively, in individual reelin-expressing cells in the postnatal cortex but not in their embryonic counterparts. We conclude that Cajal-Retzius cells express gamma-less GABA(A) receptors at embryonic stages and switch to expressing gamma-containing GABA(A) receptor isoforms during postnatal neocortical development.

Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. However, the consequence of microtubule disruption on the structure of postsynaptic GlyR/gephyrin clusters is controversial and possible alterations in function are largely unknown. In this study, we have examined the physiological and morphological properties of GlyR/gephyrin clusters after colchicine treatment in cultured spinal neurons during development. In immature neurons (5-7 DIV), disruption of microtubules resulted in a 33 ± 4% decrease in the peak amplitude and a 72 ± 15% reduction in the frequency of spontaneous glycinergic miniature postsynaptic currents (mIPSCs) recorded in whole cell mode. However, similar colchicine treatments resulted in smaller effects on 10-12 DIV neurons and no effect on mature neurons (15-17 DIV). The decrease in glycinergic mIPSC amplitude and frequency reflects postsynaptic actions of colchicine, since postsynaptic stabilization of microtubules with GTP prevented both actions and similar reductions in mIPSC frequency were obtained by modifying the Cl-driving force to obtain parallel reductions in mIPSC amplitude. Confocal microscopy revealed that colchicine reduced the average length and immunofluorescence intensity of synaptic gephyrin/GlyR clusters in immature (approximately 30%) and intermediate (approximately 15%) neurons, but not in mature clusters. Thus the structural and functional changes of postsynaptic gephyrin/GlyR clusters after colchicine treatment were tightly correlated. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic mIPSCs indicated a reorganization of the postsynaptic region from containing both α2β and α1β GlyRs in immature neurons to only α1β GlyRs in mature neurons. Microtubule disruption preferentially affected postsynaptic sites containing α2β-containing synaptic receptors.

Cajal-Retzius (CR) cells are among the earliest generated population of neurons in the developing neocortex and have been implicated in regulating cortical lamination. In rodents, CR cells are transient, being present only up to 2–3 weeks after birth. Although previous electrophysiological studies have demonstrated the presence of NMDA and GABAA receptors in CR cells, little is known about the functional properties of these receptors. Using whole-cell patch-clamp techniques in neocortical slices, we confirmed the presence of D-aminophosphonovaleric acid (APV)- and ifenprodil-sensitive NMDA receptors, and found that the functional expression of this receptor subtype is strain specific. The NMDA-induced response was consistently accompanied by overriding current transients that were blocked by APV and ifenprodil. In addition, bicuculline readily abolished these transients without affecting the NMDA-induced current response. The generation of these overriding current transients was dependent upon intracellular Ca2+ and was prevented by dialysis with the high-affinity Ca2+-chelator BAPTA. Overall, this study uncovered a synergistic interaction between these receptors, whereby activation of NMDA receptors leads to enhanced GABAA receptor-mediated activity through a Ca2+-dependent mechanism.

Despite the fact that ethanol is one of the most widely used psychoactive agents, the mechanisms and sites of action by which it modifies brain functions are only now being elucidated. Studies over the last decade have shown that ethanol can specifically alter the function of several ligand-activated ion channels including N-methyl-D-aspartate (NMDA), serotonin (5-HT(3)), glycine and GABA(A) receptors. After several years of extensive research in this field, the resolution of what, where and how ethanol modifies GABA(A) receptors continues to be controversial. For example, after demonstrating that ethanol was able to alter Cl(-) flux in synaptoneurosomes and cultured neurons, several electrophysiological studies were unable to show enhancement of the GABA(A) receptor current in single neurons. The lack of positive results with low ethanol concentrations was interpreted as being due to receptor heterogeneity and differences in intracellular modulation by protein kinases and calcium. The existence of high receptor heterogeneity with respect to ethanol sensitivity has been supported by studies done in a variety of cell types which showed that ethanol potentiated some, but not other neurons. Adding to this complexity, it was shown that while some hippocampal GABA(A) receptors can be affected by ethanol concentrations between 1 and 100 mM, others are only sensitive to concentrations above 200 mM. The curve of the relationship between low ethanol concentrations and current enhancement suggests a high degree of complexity in the molecular interaction because of its steepness and "inverted" U shape. Similarly, the effects of ethanol on GABA(A) receptors seems much more complex than those of benzodiazepines, barbiturates and neurosteroids. The major problem encountered in advancing understanding of the mechanism of ethanol action in native neuronal receptors has been the large variability detected in ethanol sensitivity. For example, several studies have shown that only some groups of neurons are sensitive to pharmacologically relevant concentrations of ethanol (1-100 mM). This receptor sensitivity variability has not been resolved using recombinant expression systems. For example, studies performed in recombinant receptors, although important for elucidating molecular requirements, have shown that they are less sensitive to ethanol suggesting that neuronal substrates are important for ethanol actions. In this review, we discuss the possibility that ethanol's action on the GABA(A) receptor may not be due solely to a direct interaction with the receptor protein, but that its effects could also be modulated by intracellular regulation, and that this latter effect is the more physiologically relevant one. Data in cortical and hippocampal neurons suggest that ethanol action on the receptor is labile, and that it also depends on repetitive stimulation and neuron integrity. In addition, the action of ethanol can be modified by activation of protein kinases and neuronal development. Finally, we discuss that the best approach for studying the interaction between the receptor and ethanol is through the combined use of recombinant receptors and overexpression in neurons.

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.

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.