Juvenile Neurons Research Articles

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABAA receptors (GABAARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABAARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg−1 day−1, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABAARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl− currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC50 or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn2+ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn2+ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABAAR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABAAR development across the brain, although the mechanism(s) underlying this action remains to be determined.

Contribution of single-channel properties to the time course and amplitude variance of quantal glycine currents recorded in rat motoneurons.

The amplitude of spontaneous, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) recorded in hypoglossal motoneurons (HMs) in an in vitro brain stem slice preparation increased over the first 3 postnatal weeks, from 42 +/- 6 pA in neonate (P0-3) to 77 +/- 11 pA in juvenile (P11-18) HMs. Additionally, mIPSC amplitude distributions were highly variable: CV 0.68 +/- 0.05 (means +/- SE) for neonates and 0.83 +/- 0.06 for juveniles. We wished to ascertain the contribution of glycine receptor (GlyR)-channel properties to this change in quantal amplitude and to the amplitude variability and time course of mIPSCs. To determine whether a postnatal increase in GlyR-channel conductance accounted for the postnatal change in quantal amplitude, the conductance of synaptic GlyR channels was determined by nonstationary variance analysis of mIPSCs. It was 48 +/- 8 pS in neonate and 46 +/- 10 pS in juvenile HMs, suggesting that developmental changes in mIPSC amplitude do not result from a postnatal alteration of GlyR-channel conductance. Next we determined the open probability (Popen) of GlyR channels in outside-out patches excised from HMs to estimate the contribution of stochastic channel behavior to quantal amplitude variability. Brief (1 ms) pulses of glycine (1 mM) elicited patch currents that closely resembled mIPSCs. The GlyR channels' Popen, calculated by nonstationary variance analysis of these currents, was approximately 0.70 (0.66 +/- 0.09 in neonates and 0.72 +/- 0.05 in juveniles). The decay rate of patch currents elicited by brief application of saturating concentrations of glycine (10 mM) increased postnatally, mimicking previously documented changes in mIPSC time course. Paired pulses of glycine (10 mM) were used to determine if rapid GlyR-channel desensitization contributed to either patch current time course or quantal amplitude variability. Because we did not observe any fast desensitization of patch currents, we believe that fast desensitization of GlyRs underlies neither phenomenon. From our analysis of glycinergic patch currents and mIPSCs, we draw three conclusions. First, channel deactivation is the primary determinant of glycinergic mIPSC time course, and postnatal changes in channel deactivation rate account for observed developmental changes in mIPSC decay rate. Second, because GlyR-channel Popen is high, differences in receptor number between synapses rather than stochastic channel behavior are likely to underlie the majority of quantal variability seen at glycinergic synapses throughout postnatal development. We estimate the number of GlyRs available at a synapse to be on average 27 in neonate neurons and 39 in juvenile neurons. Third, this change in the calculated number of GlyRs at each synapse may account for the postnatal increase in mIPSC amplitude.

Developmental aspects and mechanisms of rat caudal hypothalamic neuronal responses to hypoxia.

Previous reports from this laboratory have shown that a high percentage of neurons in the caudal hypothalamus are stimulated by hypoxia both in vivo and in vitro. This stimulation is in the form of an increase in firing frequency and significant membrane depolarization. The goal of the present study was to determine if this hypoxia-induced excitation is influenced by development. In addition, we sought to determine the mechanism by which hypoxia stimulates caudal hypothalamic neurons. Caudal hypothalamic neurons from neonatal (4-16 days) or juvenile (20-40 days) rats were patch-clamped, and the whole cell voltage and current responses to moderate (10% O2) or severe (0% O2) hypoxia were recorded in the brain slice preparation. Analysis of tissue oxygen levels demonstrated no significant difference in the levels of tissue oxygen in brain slices between the different age groups. A significantly larger input resistance, time constant and half-time to spike height was observed for neonatal neurons compared with juvenile neurons. Both moderate and severe hypoxia elicited a net inward current in a significantly larger percentage of caudal hypothalamic neurons from rats aged 20-40 days (juvenile) as compared with rats aged 4-16 days (neonatal). In contrast, there was no difference in the magnitude of the inward current response to moderate or severe hypoxia between the two age groups. Those cells that were stimulated by hypoxia demonstrated a significant decrease in input resistance during hypoxic stimulation that was not observed in those cells unaffected by hypoxia. A subset of neurons were tested independent of age for the ability to maintain the inward current response to hypoxia during synaptic blockade (11.4 mM Mg2+/0. 2 mM Ca2+). Most of the neurons tested (88.9%) maintained a hypoxic excitation during synaptic blockade, and this inward current response was unaffected by addition of 2 mM cobalt chloride to the bathing medium. In contrast, perfusion with the Na+ channel blocker, tetrodotoxin (1-2 microM) or Na+ replacement with N-methyl-D-glucamine (NMDG) significantly reduced the inward current response to hypoxia. Furthermore, the input resistance decrease observed during hypoxia was attenuated significantly during perfusion with NMDG. These results indicate the excitation elicited by hypoxia in hypothalamic neurons is age dependent. In addition, the inward current response of caudal hypothalamic neurons is not dependent on synaptic input but results from a sodium-dependent conductance.

Conditioned enhancement and suppression in the developing auditory midbrain.

Neural responses in the adult central auditory system to binaural stimuli can be altered by preceding acoustic events, including auditory motion. To determine whether the juvenile auditory system also exhibits this feature, we have examined interaural level difference (ILD) processing in the developing gerbil. A long binaural stimulus was followed without interruption by modulation of the level difference (virtual acoustic motion), which in turn was followed smoothly by a new steady state ILD. Auditory responses of single neurons in the inferior colliculus (IC) were assessed for sensitivity to the final steady state ILD. The response of EI neurons (excited by contralateral stimulation and inhibited ipsilaterally) was examined at postnatal (P) days 17-18, P24-25, and in adult animals. In adult animals, a sudden reduction of the inhibitory stimulus level resulted in a long-lasting (median = 4.3 s) enhanced discharge rate (conditioned enhancement). In P17-18 animals, conditioned enhancement only lasted for 1.2 s. When the inhibitory stimulus level was suddenly increased, adult neurons often displayed a conditioned suppression of discharge rate (median = 4.5 s), whereas P17-18 neurons remained suppressed for a much briefer period (median = 1.2 s). Moreover, the difference between conditioned responses and control discharge rates was three-four times greater in adult neurons compared to those recorded in P17-25 animals. Because conditioned responses are sensitive to the relative balance of contralateral excitation and ipsilateral inhibition, we examined the relationship between excitatory and inhibitory thresholds. In adult animals, excitatory thresholds were an average of 12 dB lower than inhibitory thresholds, while at P17-25 excitatory and inhibitory thresholds were roughly the same. These results indicate that computational properties of juvenile and adult IC neurons differ quantitatively, and this may reflect an imbalance between excitation and inhibition. The developmental differences described herein may limit the ability of young animals to locate a sound source with the latency and accuracy of an adult.

Intrinsic and synaptic properties of neurons in the vocal‐control nucleus lMAN from in vitro slice preparations of juvenile and adult zebra finches

A common theme of diverse neural systems is that circuits that are important for initial acquisition of learning do not necessarily serve as a substrate for the long-term storage of that memory. The neural basis of vocal learning in songbirds provides an example of this phenomenon, since a circuit that is necessary for vocal production during initial stages of vocal development apparently plays no subsequent role in controlling learned vocalizations. This striking functional change suggests the possibility of marked physiological changes in synaptic transmission within this circuit. We therefore examined intrinsic and synaptic properties of neurons in the cortical nucleus lMAN (lateral magnocellular nucleus of the anterior neostriatum), which forms part of this developmentally regulated circuit, in an in vitro preparation of the zebra finch forebrain. Although both functional and morphological characteristics of these neurons change substantially during vocal development, we did not observe widespread, substantive changes in the electrophysiological characteristics of juvenile versus adult lMAN neurons examined in vitro. Overall, both the intrinsic properties and synaptic responses of lMAN neurons were similar in slices from juvenile birds (at ages when lesions of lMAN disrupt vocal production) and in slices from adult birds (when lMAN lesions have no effect on song production). However, one intrinsic property that did vary between juvenile and adult cells was spike duration, which was longer in juvenile cells, suggesting the potential for activation of second-messenger cascades and/or enhanced synaptic transmission onto target cells of lMAN neurons. The pattern of synaptic response observed in both juvenile and adult cells suggests that lMAN projection neurons receive direct excitatory afferent inputs, as well as disynaptic inhibitory inputs from interneurons within lMAN. Activation of inhibitory interneurons rapidly curtails the excitatory response seen in projection neurons. This inhibition was abolished by bicuculline, indicating that the inhibitory interneurons normally exert their postsynaptic response via GABAA receptors on projection neurons. The inhibitory response could also be blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), suggesting that the activation of inhibitory interneurons within lMAN may be governed primarily by AMPA receptors. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 642–658, 1998

Intrinsic and synaptic properties of neurons in the vocal-control nucleus lMAN fromin vitro slice preparations of juvenile and adult zebra finches

A common theme of diverse neural systems is that circuits that are important for initial acquisition of learning do not necessarily serve as a substrate for the long-term storage of that memory. The neural basis of vocal learning in songbirds provides an example of this phenomenon, since a circuit that is necessary for vocal production during initial stages of vocal development apparently plays no subsequent role in controlling learned vocalizations. This striking functional change suggests the possibility of marked physiological changes in synaptic transmission within this circuit. We therefore examined intrinsic and synaptic properties of neurons in the cortical nucleus IMAN (lateral magnocellular nucleus of the anterior neostriatum), which forms part of this developmentally regulated circuit, in an in vitro preparation of the zebra finch forebrain. Although both functional and morphological characteristics of these neurons change substantially during vocal development, we did not observe widespread, substantive changes in the electrophysiological characteristics of juvenile versus adult IMAN neurons examined in vitro. Overall, both the intrinsic properties and synaptic responses of IMAN neurons were similar in slices from juvenile birds (at ages when lesions of IMAN disrupt vocal production) and in slices from adult birds (when IMAN lesions have no effect on song production). However, one intrinsic property that did vary between juvenile and adult cells was spike duration, which was longer in juvenile cells, suggesting the potential for activation of second-messenger cascades and/or enhanced synaptic transmission onto target cells of IMAN neurons. The pattern of synaptic response observed in both juvenile and adult cells suggests that IMAN projection neurons receive direct excitatory afferent inputs, as well as disynaptic inhibitory inputs from interneurons within IMAN. Activation of inhibitory interneurons rapidly curtails the excitatory response seen in projection neurons. This inhibition was abolished by bicuculline, indicating that the inhibitory interneurons normally exert their postsynaptic response via GABA(A) receptors on projection neurons. The inhibitory response could also be blocked by CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), suggesting that the activation of inhibitory interneurons within IMAN may be governed primarily by AMPA receptors.

Anterior Forebrain Neurons Develop Selectivity by an Intermediate Stage of Birdsong Learning

Auditory neurons of the anterior forebrain (AF) in adult zebra finches are highly selective for the bird's own song (BOS): they respond more to BOS than to songs of other zebra finches (conspecifics) and to BOS played in reverse. In contrast, juvenile AF neurons are not selective at 30 d of age, responding equally well to all song stimuli. Both BOS and tutor song experience are required by juveniles for normal song learning and may produce the selective properties of adult neurons. Because such selectivity could subserve song learning, it is important to determine when it arises. Birds were therefore studied at an intermediate stage of learning, after substantial experience of both tutor song and their own developing (plastic) song. Extracellular single neuron recordings in 60-d-old zebra finches revealed that AF neurons had significant song and order selectivity for both tutor song and BOS (the bird's plastic song). The degree of BOS selectivity was less than that found in adults, as indicated in part by 60 d neurons that were sensitive to the local order within syllables but not yet to the global order of syllables within a song. When responses to BOS and tutor song were compared, most neurons preferred BOS, some preferred tutor song, and others responded equally to both stimuli. The latter type of neuron was not simply immature, because many of these neurons responded significantly more to BOS and tutor song than to conspecific and reverse songs. The selectivity of AF neurons at 60 d is markedly different from the unselective properties of neurons at 30 d and may function in vocal learning at this stage. Moreover, the selectivity for both BOS and tutor song raises the possibility that both aspects of the birds' sensory experience during learning are reflected in properties of AF neurons.

Song- and Order-Selective Neurons in the Songbird Anterior Forebrain and their Emergence during Vocal Development

Auditory experience is critical for vocal learning in songbirds as in humans. Therefore, in a search for neural mechanisms for song learning and recognition, the auditory response properties of neurons in the anterior forebrain (AF) pathway of the songbird brain were investigated. This pathway plays an essential but poorly understood role during the period of song development when auditory feedback is most crucial. Single-unit recordings demonstrated that both the lateral magnocellular nucleus of the anterior neostriatum (LMAN) and Area X (X) contain auditory neurons in adult male finches. These neurons are strongly selective for both spectral and temporal properties of song; they respond more robustly to the bird's own song (BOS) than to songs of conspecific individuals, and they respond less well to the BOS if it is played in reverse. In addition, X neurons are more broadly responsive than LMAN neurons, suggesting that responses to song become progressively more refined along this pathway. Both X and LMAN of young male finches early in the process of song learning (30-45 d old) also contain song-responsive auditory neurons, but these juvenile neurons lack the song and order selectivity present in adult birds. The spectral and temporal selectivity of the adult AF auditory neurons therefore arises during development in neurons that are initially broadly song-responsive. These neurons provide one of the clearest examples of experience-dependent acquisition of complex stimulus selectivity. Moreover, the auditory properties of the AF circuit suggest that one of its functions may be to mediate the auditory learning and feedback so essential to song development.

Ionic Currents Underlying Developmental Regulation of Repetitive Firing inAplysiaBag Cell Neurons

We have investigated the developmental regulation of the ability to fire repetitively in the bag cell neurons of Aplysia californica, a neuronal system in which the behavioral effects of repetitive firing are well characterized. Adult bag cell neurons exhibit an afterdischarge, consisting of prolonged depolarization and repetitive firing, which causes the release of several peptides from these neurons that induce egg-laying behaviors. Afterdischarge can be triggered in vitro by a variety of stimuli, including electrical stimulation and exposure to the potassium channel blocker tetraethyl ammonium chloride (TEA). In contrast to adults, juvenile neurons did not exhibit afterdischarge in response to pleural-abdominal connective shock or TEA. Juvenile neurons did exhibit, however, prolonged depolarizations in the presence of TEA, perhaps reflecting the anlage of the mechanism responsible for afterdischarge in the adult. To investigate developmental mechanisms underlying the regulation of repetitive firing, we compared ionic currents in adult and juvenile bag cell neurons. We found that during the period in which these neurons acquire the capacity to fire repetitively, a number of currents are regulated: (1) three K+ currents decrease (Ca2+)-dependent K+ and two components of voltage-dependent delayed-rectifier K+ current); (2) A-type K+ current increases; and (3) two Ca2+ currents increase (basal and PKC-activated). This pattern is consistent with the increase in the ability to fire repetitively that we observe during maturation: our results indicate that developmental control of repetitive firing in this system is accompanied by selective regulation of specific ionic currents which, after maturation, play important roles in generating the afterdischarge and triggering egg-laying behaviors.

P-10-28 - Anticonvulsant and anxiolytic effects of LY300164, an orally effective AMPA antagonist

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. Kainate receptors (KARs) play an important role in sensory transmission. Five different KAR subunits have been cloned and the expression of the KAR subunits showed developmental changes. In this study, RT-PCR, western blotting, immunohistochemistry and a patch clamp technique were used examine the functional expression of the GluR5 subunit in the SG of the Vc in juvenile, peripubertal and/or adult mice. The levels of mRNA and protein expression of the GluR5 subunit in the SG of the Vc were higher in the juvenile mice than in the peripubertal or adult mice. In addition, the KA and ATPA, a GluR5 KAR agonist, induced membrane depolarization on the SG neurons in both juvenile and adult mice in a concentration-dependent manner. However, the juvenile SG neurons showed a stronger response to KA and ATPA than those of adults. The membrane depolarization by KA was suppressed slightly in the presence of the AMPA receptor antagonist, GYKI 52466. These results show that the GluR5 KAR subunits are expressed functionally on the SG neurons of the Vc in mice, and the expression levels of the GluR5 subunits decrease with postnatal development. These postnatal changes in the GluR5 KAR subunit may be a possible mechanism for age-dependent pain processing.

Three distinct neuronal phenotypes exist in embryonic rat hippocampal neurons cultured in basic fibroblast growth factor

The possibility that neurons cultured in basic fibroblast growth factor (bFGF) are heterogeneous raises concerns about their subsequent use in gene transfection and transplantation studies. We have examined the fate of embryonic hippocampal neurons in bFGF culture, and now conclude that these cells are not only heterogeneous, but possess neurons of various stages of development. Morphological and immunocytochemical analysis reveal three distinct developmental classes of neurons are present in extended bFGF culture. This triparticle classification is supported by electrophysiological analysis, which reveals that upon depolarization, neurons with precursor and juvenile neuron morphologies are unable to fire action potentials. The third class of neurons, which resemble age-matched polarizes neurons in standard serum culture, fired multiple action potentials indicative of a mature phenotype. These data show neurons at multiple developmental stages co-exist in bFGF culture, and provide an experimental basis for their classification.

Differential modulatory actions of serotonin in Aplysia sensory neurons: implications for development and learning

We have addressed whether mechanisms of adult plasticity reflect conserved forms of developmental processes by comparing the effects of serotonin (5HT) in juvenile and adult Aplysia sensory neurons. We show that the effects of 5HT can be dissociated into two functional classes which (i) contribute differentially to short- and long-term synaptic plasticity in adults and (ii) emerge differentially during development. We propose a model in which one class of mechanism mediates structural changes early in development and is retained in the adult to subserve long-term memory, while the second class develops late and contributes only to short-term memory in the adult.

Chapter 23 Formation of chemical synapses by adult Aplysia neurons in vitro is facilitated by the presence of juvenile neurons

Publisher Summary This chapter describes that L10 and target cells L2–L6, isolated from adult ganglia and cocultured under the same in vitro conditions as their juvenile counterparts, show a marked reduction in forming chemical synapses. The adult cells, however, can form chemical connections indistinguishable from those formed by juvenile neurons when they are cocultured with juvenile neurons. The methods used for the isolation and maintenance of Aplysia neurons in culture have been described in the chapter. During development, synapse formation between a neuron and its specific target occurs via a multistep process that includes axon growth toward the appropriate region, the formation and stabilization of appropriate connections, and the elimination of inappropriate connections. The chapter provides results on synapse formation between juvenile cells and adult cells in vitro . The results indicate that synapse formation among adult cells is significantly reduced when cultured alone. When they are plated in the presence of juvenile neurons, either target cells of L10 (left upper quadrant cells) or nontarget cells (right upper quadrant cells), synapse formation between adult L10 and adult left upper quadrant cells is enhanced.

The postnatal development of visual callosal connections in the absence of visual experience or of the eyes.

Counts of callosal neurons retrogradely labeled by horseradish peroxidase (visualized using multiple substrates) were obtained in areas 17 and 18 of five kittens reared with their eyelids bilaterally sutured and of three kittens which had undergone bilateral enucleation on postnatal days 1--4. These counts were compared with those obtained in normal adult cats. The normal adult distribution of the callosal neurons results from the gradual postnatal reduction of a more widespread juvenile population. Binocular visual deprivation by lid suturing dramatically decreases the final number of callosal neurons and narrows their region of distribution (callosal zone) in areas 17 and 18. A less severe reduction in the final number of callosal neurons is caused by bilateral enucleation, which also increases the width of the callosal zone compared to that of normal cats. Thus, visual experience is necessary for the normal stabilization of juvenile callosal connections. However, since some callosal neurons form connections in the absence of vision, other influences capable of stabilizing juvenile callosal neurons also exist. These influences are probably antagonized by destabilizing influences or inhibited, when the eyes are intact.