Distinct developmental trajectories of autonomous behaviors and agency in rodents.

Behavioral interactions within the nuclear family may play a pivotal role in the emergence of autonomy and agency in mammals. While the emergence of a behavior may arise over weeks in line with nervous system maturation, individual events occur on sub-second time scales. This makes it uniquely challenging to track development in the lab where observations are made over minutes to hours or in ecological studies which lack individual specificity and sub-second precision. Here we study families of gerbils, a highly social rodent, raised in enlarged home-cage environments over weeks of development, using continuous video recordings to capture tens of millions of time points per family. Focusing on postnatal day 15 (when pups leave the nest) to day 30 (around the time when pups would disperse) we identify distinct developmental trajectories for both autonomous behaviors (exploration, food and water foraging), and social behaviors (huddling, approach, time spent together). Most of these behaviors emerge in concert with clear diurnal and crepuscular patterns and we find sex differences in both autonomous and social behaviors. Our work supports the emergence of distinct autonomous and social behavior phenotypes as the behavioral correlates of critical developmental periods of maturation of the rodent brain and can form the basis of future research on development from both neuroscience and behavioral biology perspectives.

GABA(B) and Trk receptor signaling mediates long-lasting inhibitory synaptic depression.

In many areas of the nervous system, excitatory and inhibitory synapses are reconfigured during early development. We have previously described the anatomical refinement of an inhibitory projection from the medial nucleus of the trapezoid body to the lateral superior olive in the developing gerbil auditory brain stem. Furthermore, these inhibitory synapses display an age-dependent form of long-lasting depression when activated at a low rate, suggesting that this process could support inhibitory synaptic refinement. Since the inhibitory synapses release both glycine and GABA during maturation, we tested whether GABA(B) receptor signaling could initiate the decrease in synaptic strength. When whole cell recordings were made from lateral superior olive neurons in a brain slice preparation, the long-lasting depression of medial nucleus of the trapezoid body-evoked inhibitory potentials was eliminated by the GABA(B) receptor antagonist, SCH-50911. In addition, inhibitory potentials could be depressed by repeated exposure to the GABA(B) receptor agonist, baclofen. Since GABA(B) receptor signaling may not account entirely for inhibitory synaptic depression, we examined the influence of neurotrophin signaling pathways located in the developing superior olive. Bath application of brain-derived neurotrophic factor or neurotrophin-3 depressed evoked inhibitory potentials, and use-dependent depression was blocked by the tyrosine kinase antagonist, K-252a. We suggest that early expression of GABAergic and neurotrophin signaling mediates inhibitory synaptic plasticity, and this mechanism may support the anatomical refinement of inhibitory connections.

Development and influence of inhibition in the lateral superior olivary nucleus.

While studies of neuronal development and plasticity have focused on excitatory pathways, the inhibitory projection from the MNTB to the LSO provides a favorable model for studies of synaptic inhibition. This review covers recent studies from our laboratories indicating that inhibitory connections are quite dynamic during development. These findings suggest that there are two phases inhibitory transmission. During an initial depolarizing phase is growth and branching of pre- and postsynaptic elements in the LSO. During a second hyperpolarizing phase there is refinement of inhibitory afferent arborizations and the LSO dendrites that they innervate.

Long-lasting inhibitory synaptic depression is age- and calcium-dependent.

The developmental refinement of excitatory synapses is often influenced by neuronal activity, and underlying synaptic mechanisms have been suggested. In contrast, few studies have asked whether inhibitory synapses are reorganized during development and whether this is accompanied by use-dependent changes of inhibitory synaptic strength. The topographic inhibitory projection from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) undergoes synapse elimination during development (Sanes and Takács, 1993). To determine whether there is an associated period of synaptic plasticity, whole-cell recordings were obtained from developing LSO neurons of gerbils in a brain slice preparation. In current-clamp recordings, low-frequency stimulation of the MNTB led to a decline in IPSP amplitude by 43%. In voltage-clamp recordings, hyperpolarized LSO neurons also exhibited a long-lasting depression of MNTB-evoked inhibitory synaptic currents (34%) after low-frequency stimulation. When LSO neurons were depolarized, low-frequency stimulation of the MNTB produced a significantly larger inhibitory synaptic depression (59%). This synaptic plasticity declined dramatically by postnatal days 17-19. Similar to well studied forms of excitatory synaptic plasticity, inhibitory depression depended on postsynaptic calcium. We propose that such activity-dependent synaptic depression may support the developmental rearrangement of inhibitory terminals as they compete with neighboring excitatory and/or inhibitory inputs.

Afferent regulation of inhibitory synaptic transmission in the developing auditory midbrain.

To determine whether afferent innervation regulates the strength of inhibitory connections in the gerbil auditory midbrain, both cochleas were surgically removed in postnatal day 7 animals, before sound-driven activity is first observed. Inhibitory synaptic currents were measured in a brain slice preparation 1-7 d after the ablations. Whole-cell and gramicidin-perforated patch recordings were obtained from inferior colliculus neurons, and IPSCs were evoked by stimulation of the commissure of the inferior colliculus (CIC) or the ipsilateral lateral lemniscus (LL) in the presence of kynurenic acid. Deafferentation led to a 24 mV depolarizing shift in the IPSC equilibrium potential within 1 d of deafferentation. As a consequence, there was a large reduction of IPSC amplitude at a holding potential of -20 mV in neurons from bilaterally ablated animals. Furthermore, both afferent pathways displayed a 50% reduction of the inhibitory synaptic conductance after deafferentation, indicating that driving force was not solely responsible for the decline in IPSC amplitude. When paired pulses were delivered to the LL or CIC pathway in control neurons, the evoked IPSCs exhibited facilitation. However, paired pulse facilitation was nearly eliminated after deafferentation. Thus, normal innervation affects inhibitory synaptic strength by regulating postsynaptic chloride homeostasis and presynaptic transmitter release properties.

Age-dependent failure of axon regeneration in organotypic culture of gerbil auditory midbrain.

Inferior colliculus (IC) slice cultures from postnatal (P) day 6-8 gerbils exhibit axonal regeneration across a lesion site, and these regrowing processes can form synapses. To determine whether regenerative capacity is lost in older tissue, as occurs in vivo, slices from P12-21-day animals were grown under similar conditions. While these cultures displayed a near complete loss of neurons over 6 days in vitro, glutamate receptor antagonists (AP5 and/or CNQX) significantly enhanced survival, particularly at P12-15. In contrast, several growth factors or high potassium did not improve neuron survival. Therefore, axonal regeneration was assessed following complete transection of the commissure in AP5/CNQX-treated IC cultures from P12 animals. Neurofilament staining revealed that transected commissural axons survived for 6 days in vitro, but only a few processes crossed the lesion site and these axons did not extend into the contralateral lobe. In contrast, there was robust axonal sprouting and growth within one lobe of the IC, remote from the lesion site. When P6 and P12 tissue was explanted onto a coated substrate, the P6 axons grew onto the substrate, but the P12 axons were seemingly prevented from reaching the substrate by a veil of nonneuronal cells. Coculture of the IC and one of its afferent populations, the lateral superior olive, provided a similar finding, indicating that failure to regenerate was a general property at the age examined. These data show that neuron survival is not sufficient to permit axon regeneration at P12, and suggest that P12 lesion sites manufacture a prohibitive substrate since process outgrowth is blocked specifically at the commissure transection.

Serotonergic modulation of synapses in the developing gerbil lateral superior olive.

The lateral superior olive (LSO) is a primary site of binaural convergence that responds selectively to changes in interaural level difference (ILD) by integrating ipsilateral excitatory and contralateral inhibitory inputs. The circuit matures during the first three postnatal weeks, undergoing several structural and functional changes that are influenced by afferent activity. Therefore modulation of synaptic activity by neuromodulators may participate in the maturation of this circuit. The present study describes robust effects of serotonin (5-HT) on LSO synaptic function. Using whole cell voltage-clamp recording from gerbil LSO neurons (postnatal days 6-13) in an in vitro slice preparation, we have identified several distinct forms of serotonergic modulation of spontaneous and evoked synaptic transmission. First, 1-2 min application of 5-HT (100 microM) activated prolonged bursts of spontaneous inhibitory postsynaptic currents (IPSCs). However, there was an age-dependent decline, such that this effect rarely was observed beyond postnatal day 8. 5-HT apparently increased the excitability of inhibitory afferents, because 5-HT-induced IPSCs were blocked by tetrodotoxin. A second effect of 5-HT was to depress rapidly and profoundly the amplitude of electrically evoked excitatory postsynaptic currents (EPSCs). In contrast, 5-HT also depressed evoked IPSCs but to a significantly lesser degree. The receptor subtypes mediating these effects were examined using specific 5-HT agonists and antagonists. A 5-HT1 agonist, 5-carboxamidotryptamine, produced EPSC depression but did not induce spontaneous IPSCs. A 5-HT2 agonist, alpha-Me-5-HT, reproduced all the observed effects of 5-HT (PSC depression as well as induction of spontaneous IPSCs), whereas a 5-HT2 antagonist, ketanserin, blocked the induction of spontaneous IPSCs. Therefore induction of spontaneous IPSCs is mediated by 5-HT2 receptors, whereas both 5-HT1 and 5-HT2 receptor types contribute to PSC depression. Serotonergic modulation of LSO synapses may have consequences for both developmental plasticity and auditory function. Serotonergic induction of IPSCs was observed primarily in young animals and thus may represent a mechanism for amplifying the activity of inhibitory synapses in LSO during a period of use-dependent plasticity in postnatal development. PSC depression, which preferentially affects excitation, is a potential mechanism for modulation of ILD tuning.

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.

Commissural and lemniscal synaptic input to the gerbil inferior colliculus.

The central nucleus of the inferior colliculus (ICC) receives direct inputs, bilaterally, from all auditory brain stem nuclear groups. To evaluate the contribution made to gerbil ICC neuron physiology by two major afferent pathways, we examined the synaptic responses evoked by direct stimulation of the commissure of the inferior colliculus (CIC) and the ipsilateral lateral lemniscus (LL). Frontal midbrain slices were obtained from postnatal day (P) 9-P19 gerbils, and whole cell recordings were made under current- (n = 22) or voltage-clamp (n = 52) conditions. Excitatory and inhibitory synaptic responses were characterized by sequentially exposing the slice to ionotropic glutamate receptor antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) + aminophosphonpentanoic acid (AP-5), or kynurenic acid)], a gamma-aminobutryic acid type A receptor antagonist (bicuculline), and a glycine receptor antagonist (strychnine). In current clamp, LL stimulation typically produced a short latency depolarization followed by a longer duration hyperpolarization. The depolarization was abolished by AP-5 + CNQX, and the remaining inhibitory potential displayed either bicuculline or strychnine sensitivity. In voltage clamp, 79% of ICC neurons displayed synaptic currents after stimulation of each pathway. The synaptic currents were typically complex waveforms, and ionotropic glutamate receptor antagonists reduced inward currents at a holding potential of -80 mV in the majority of neurons. In addition, this treatment reduced outward synaptic currents at a holding potential of -20 mV, indicating that inhibitory interneuronal input was often activated by LL or CIC afferents. A minority of neurons had synaptic currents that were unaffected by glutamate receptor antagonists, but it was more common for CIC-evoked currents to be unaffected (38%) rather than LL-evoked currents (22%). The CIC provided a strong inhibitory input that was almost exclusively GABAergic, whereas the LL inhibition often included a glycinergic component. These experiments have shown that the CIC provides a major glutamatergic and GABAergic input to most ICC neurons. However, much of the inhibitory input from both the CIC and the LL appears to be mediated by interneuronal connections.

Metabotropic glutamate receptor activation modulates sound level processing in the cochlear nucleus.

The principal role of ionotropic glutamate receptors in the transmission and processing of information in the auditory pathway has been investigated extensively. In contrast, little is known about the functional contribution of the G-protein-coupled metabotropic glutamate receptors (mGluRs), although their anatomic location suggests that they exercise a significant influence on auditory processing. To investigate this issue, sound-evoked responses were obtained from single auditory neurons in the cochlear nuclear complex of anesthetized cats and gerbils, and metabotropic ligands were administered locally through microionophoretic pipettes. In general, microionophoresis of the mGluR agonists, (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid or (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine, initially produced a gradual increase in spontaneous and sound-evoked discharge rates. However, activation and recovery times were significantly longer than those observed for ionotropic agonists, such as N-methyl--aspartate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, consistent with the recruitment of a second-messenger system. The efficacy of mGluR agonists was diminished after administration of the mGluR antagonist, (+)-alpha-methyl-4-carboxyphenylglycine, consistent with a selective action at metabotropic recognition sites. In contrast, two distinct changes were observed after the mGluR agonist had been discontinued for several minutes. Approximately 50% of neurons exhibited a chronic depression of sound-evoked discharge rate reminiscent of long-term depression, a cellular property observed in other systems. Approximately 30% of neurons exhibited a long-lasting enhancement of the sound-evoked response similar to the cellular phenomenon of long-term potentiation. These findings suggest that mGluR activation has a profound influence on the gain of primary afferent driven activity in the caudal cochlear nucleus.

A developmental shift from GABAergic to glycinergic transmission in the central auditory system.

GABAergic and glycinergic circuits are found throughout the auditory brainstem, and it is generally assumed that transmitter phenotype is established early in development. The present study documents a profound transition from GABAergic to glycinergic transmission in the gerbil lateral superior olive (LSO) during the first 2 postnatal weeks. Whole-cell voltage-clamp recordings were obtained from LSO neurons in a brain slice preparation, and IPSCs were evoked by electrical stimulation of the medial nucleus of the trapezoid body (MNTB), a known glycinergic projection in adult animals. GABAergic and glycinergic components were identified by blocking transmission with bicuculline and strychnine (SN), respectively. In the medial limb of LSO, there was a dramatic change in the GABAergic IPSC component, decreasing from 78% at postnatal day 3 (P3)-P5 to 12% at P12-P16. There was an equal and opposite increase in the glycinergic component during this same period. Direct application of GABA also elicited significantly larger amplitude and longer duration responses in P3-P5 neurons compared with glycine-evoked responses. In contrast, MNTB-evoked IPSCs in lateral limb neurons were more sensitive to SN throughout development. Consistent with the electrophysiological observations, there was a reduction in staining for the beta2,3-GABAA receptor subunit from P4 to P14, whereas staining for the glycine receptor-associated protein gephyrin increased. Brief exposure to baclofen depressed transmission at excitatory and inhibitory synapses for approximately 15 min, suggesting a GABAB-mediated metabotropic signal. Collectively, these data demonstrate a striking switch from GABAergic to glycinergic transmission during postnatal development. Although GABA and glycine elicit similar postsynaptic ionotropic responses, our results raise the possibility that GABAergic transmission in neonates may play a developmental role distinct from that of glycine.

Afferent regulation of glycine receptor distribution in the gerbil LSO.

Synaptic activity plays an important role in many aspects ofneuronal development, particularly the expression of proteins. In this study, the influence of inhibitory and excitatory afferents on the development of glycine receptor density in the lateral superior olive (LSO) of Mongolian gerbils was investigated. Afferent activity was manipulated by removing one or both cochleas at postnatal day 7, prior to the onset of sound-evoked responses. Due to the anatomy of the LSO, these manipulations result in either excitatory denervation, inhibitory denervation, or both. The density of glycine receptors in the LSO was determined at 21 days postnatal. Glycine receptors were either labeled with tritiated strychnine (3H-SN) or with an antibody directed against gephyrin, a protein closely associated with the receptor complex. Antibody binding was used to quantify the differential glycine receptor density between the medial limb (high frequency area) and the lateral limb (low frequency area) of the LSO. 3H-SN was used to quantify the amount of glycine receptors in each part of the LSO in control and experimental animals. In addition, changes in neuron density and neuron cross-sectional area were quantified following cochlear ablations. In control animals, the amount of glycine receptors is about 2- to 3-fold higher in the high-frequency than in the low-frequency region. In bilaterally ablated animals, the same density of glycine receptors was measured in the high- and low-frequency region. Unilateral ablations had no significant effect on glycine receptor distribution, either ipsi- or contralateral to the ablation. The neuron cross-sectional area decreased about 30% in the ipsilateral LSO of unilaterally ablated animals and in bilaterally ablated animals. However, alterations of soma density and cross-sectional area were similar in the high- and low-frequency projection region. These results suggest that the distribution of glycine receptors is only changed when excitatory and inhibitory afferents have been denervated.

Modulation of calcium by inhibitory systems in the developing auditory midbrain.

Inhibitory synaptic transmission is of fundamental importance during the maturation of central auditory circuits, and their subsequent ability to process acoustic information. The present study investigated the manner in which inhibitory transmission regulates intracellular free calcium levels in the gerbil inferior colliculus using a brain slice preparation. Inhibitory and excitatory postsynaptic potentials were evoked by electrical stimulation of the ascending afferents at the level of the dorsal nucleus of the lateral lemniscus. Pharmacologically isolated inhibitory synaptic potentials were able to attenuate a calcium rise in collicular neurons that was generated by depolarizing current injection. In addition, GABA(A) and glycine receptor antagonists typically led to an increase of calcium in collicular neurons during electrical stimulation of the ascending afferent pathway at the level of the dorsal nucleus of the lateral lemniscus. Bath application of GABA or muscimol, a GABA(A) receptor agonist, evoked a brief hyperpolarization followed by a long-lasting depolarization in inferior colliculus neurons. This treatment also induced a transient calcium increase that correlated with the membrane depolarization phase. Baclofen, a GABA(B) receptor agonist, had no effect on either membrane potential or calcium levels. Ratiometric measures indicated that the muscimol-evoked rise in calcium was approximately 150 nM above basal levels. The muscimol-evoked responses were completely antagonized by bicuculline and attenuated by picrotoxin. Together, these results suggest that inhibitory synaptic transmission participates in the regulation of postsynaptic calcium during the developmental period. Inhibitory transmission may attenuate a calcium influx that is evoked by excitatory synapses, but it can also produce a modest influx of calcium when activated alone. These mechanisms may help to explain the influence of inhibitory transmission on the development of postsynaptic properties.

Role of synaptic inhibition in processing of dynamic binaural level stimuli.

We have recently discovered a paradoxical aftereffect associated with inhibition in the gerbil auditory midbrain. Single neurons in the inferior colliculus (IC) were assessed for sensitivity to a virtual motion stimulus produced by modulating the interaural level difference (ILD), a major cue for sound localization. The class of neuron studied was predominantly excited by contralateral stimulation and inhibited by ipsilateral stimulation. Sound pressure level was modulated trapezoidally at the ipsilateral "inhibitory" ear, whereas the contralateral "excitatory" level remained constant. When the inhibitory stimulus was decreased within a range of sound levels that maintained suppression under static conditions, an unexpected discharge was often elicited, apparently because of an aftereffect of synaptic inhibition. In contrast, when the inhibitory stimulus was increased within a range of sound levels that produced only modest suppression under static conditions, neuronal discharge was often profoundly suppressed. In many cases the "conditioned enhancement" or "conditioned suppression" persisted for several seconds after the modulation of ILD, and such conditioned responses were influenced by the modulation depth and rate. To test the effect of inhibition in the IC directly, glycine and GABA were pulsed from a glass recording pipette during a constant monaural excitatory stimulus. The acoustically elicited discharge rate was potentiated markedly if preceded immediately by the brief (0.5-10 sec) application of inhibitory transmitter. Collectively, these results revealed unusually long-lasting effects of inhibition that may establish a new range of acoustic cues to which the neuron responds best. This may have broad implications for processing ensuing auditory stimuli.

In vitro induction of microcyst-like structures in the superior olivary complex.

To investigate the etiology of hole formation in the gerbil and rat central auditory system, organotypic cultures were grown in control and veratridine-containing media. The latter condition is known to increase neuronal activity. Tissue was obtained at postnatal day 6 and grown for 6-9 days in vitro, a period prior to the formation of holes in vivo. In both rats and gerbils, veratridine led to the appearance of large numbers of holes, and these were phenotypically similar to those found in vivo. These results support the idea that hole formation is an activity-dependent phenomenon, and suggest that it is not restricted to the mature gerbil auditory system.

Deafferentation weakens excitatory synapses in the developing central auditory system.

Decreased excitatory synaptic activity during development often leads to pre- and postsynaptic atrophy, as assessed anatomically. The present study considers the effect of decreased excitatory transmission on the maturation of synaptic strength. Towards this end, cochlear nucleus neurons, which project to the ipsilateral lateral superior olive (LSO), were denervated in gerbils at postnatal day 7, before the onset of hearing. This manipulation was intended to disrupt spontaneous glutamatergic transmission in the LSO while sparing the glycinergic afferents from the medial nucleus of the trapezoid body (MNTB). Afferent-evoked synaptic activity was assessed 1-6 days after ablation in a brain slice preparation using whole-cell current- and voltage-clamp recordings. In control animals, ipsilaterally evoked excitatory postsynaptic potentials (EPSPs) were present in 91% of neurons tested, but were observed in only 60% of neurons following cochlea removal. The maximum EPSP amplitude was significantly smaller in manipulated neurons compared with controls, and this was accompanied by a higher incidence of ipsilaterally evoked inhibitory postsynaptic potentials (IPSPs). To study the efficacy of excitatory synapses in greater detail, voltage-clamp recordings were made in the presence of strychnine and AP-5 [D(O)-2-amino-5-phosphonopentanoic acid]. The minimum excitatory postsynaptic current (EPSC) amplitude, presumed to reflect the efficacy of a single glutamatergic afferent, was approximately 40% smaller in manipulated neurons. In contrast, MNTB-evoked IPSPs were similar in neurons from control and ablated animals. However, manipulated neurons often exhibited a rebound depolarization after a hyperpolarizing current pulse or an afferent-evoked IPSP. In 70% of manipulated neurons, synaptically evoked rebound depolarizations were reduced, but not eliminated, by glutamate receptor antagonists. The glycine receptor antagonist strychnine did eliminate the IPSP-associated depolarization in these neurons. Collectively, these results suggest that functional denervation of excitatory afferents decreases their synaptic efficacy as result of both cell loss as well as decreased strength of individual surviving synapses.

Glycinergic transmission regulates dendrite size in organotypic culture.

We previously demonstrated that inhibitory synaptic transmission influences dendrite development in vivo. We now report an analogous finding in an organotypic culture of a glycinergic projection nucleus, the medial nucleus of the trapezoid body (MNTB), and its postsynaptic target, the lateral superior olive (LSO) of gerbils. Cultures were generated at 6-7 days postnatal and grown in serum containing medium with or without the glycine receptor antagonist, strychnine (SN), at 2 microM. LSO neurons were then labeled with biocytin, and the dendritic arbors were analyzed morphometrically. Compared to neurons form age-matched in vivo tissue, the neurons cultured in control media were somewhat atrophic, including decreases in dendritic branching and length. Incubation in strychnine led to a dramatic increase in dendritic branching and total dendritic length. Control neurons averaged 6.3 branches compared to 18 branches/neuron in SN-treated cultures. There was a similar increase in primary dendrites and total dendritic length. The physical elimination of MNTB cells did not mimic SN treatment, presumably because glycinergic LSO neurons generated intrinsic connections. In fact, the LSO soma area was significantly greater following MNTB removal, suggesting that these afferents provide a second signal to postsynaptic neurons. These results suggest that spontaneous glycinergic transmission regulates the growth of postsynaptic processes.

Differential expression of MAG, MBP and L1 in the developing lateral superior olive.

The aim of this study was to investigate whether glial-associated molecules exhibit a pattern of expression that could influence oriented dendrite outgrowth in the gerbil lateral superior olive (LSO). In particular, we have previously noted that axon fascicles are oriented parallel to isofrequency laminae in the medial limb of the LSO, as are LSO dendrites, a phenotype that emerges postnatally. Therefore, we examined the immunocytochemical staining pattern of antibodies directed against three proteins that are found along axons: myelin basic protein (MBP), myelin-associated glycoprotein (MAG), and neuron-glia cell adhesion molecule (L1). MAG staining was first observed at postnatal day (P) 4 on the axon fibers surrounding the LSO. By P7 there was a differential pattern of MAG staining within the LSO, and immunopositive fibers were observed solely in the medial limb (e.g., high frequency projection region). Between P7 and P12, MAG staining was restricted largely to fascicles in the medial limb, and these were oriented parallel to the isofrequency axes. Few positive fibers of irregular orientation were observed in the lateral limb (e.g., low frequency projection region). Significant MAG-staining was not observed in the lateral limb until P15. The MAG immunoreactivity extended throughout the LSO by P21, although it was no longer restricted to axon fascicles. In contrast, MBP-positive fibers were uniformly distributed within the LSO by P12. Finally, L1 was found on oriented axon fascicles at P0, but became sparsely distributed throughout the LSO neuropil after P7, and was restricted to neuron cell bodies in the adult. Taken together, the results suggest that oriented axon fascicles bearing MAG and L1 may contribute to the developmental refinement of dendrite and axon arbors within the LSO.

Regional distribution of neurotrophin receptors in the developing auditory brainstem.

Neuron survival and axonal regeneration become severely limited during early postnatal development. In conjunction with our recent organotypic analysis of regeneration in the auditory midbrain, we wished to determine whether neurotrophins could serve as a trophic substance during the postnatal period. Therefore, the current study examines the development of three neurotrophin receptor tyrosine kinases (TrkA, TrkB, and TrkC) in the gerbil auditory brainstem. Immunoreactivity to TrkA, the nerve growth-factor receptor, was observed in nonneuronal cells during the first two postnatal weeks. In the cochlear nucleus of mature animals, however, there was a TrkA-positive neuronal subpopulation. In contrast, immunoreactivity to TrkB and TrkC (the receptors for brain-derived neurotrophic factor and neurotrophin-3, respectively) displayed a widespread distribution in the auditory brainstem. At postnatal day 0, TrkB and TrkC staining was virtually absent from auditory nuclei, although immunopositive neurons were present in the mesencephalic trigeminal nucleus. By postnatal day 7, TrkB- and TrkC-positive neurons were present in most brainstem auditory nuclei. At postnatal day 15, TrkB immunoreactivity was observed throughout the inferior colliculus (IC), the cochlear nucleus, the medial and lateral nuclei of the trapezoid body, and the lateral superior olive, whereas TrkC labeled only a subpopulation of neurons within the central nucleus of the IC. The TrkB immunoreactivity was present on both neuronal somata and dendrites, whereas TrkC was generally restricted to cell bodies. At postnatal day 30, TrkB immunostaining was observed on most neurons of the IC. The medial and lateral nuclei of the trapezoid body displayed extremely strong TrkB staining, followed by the cochlear nucleus. In contrast, the TrkC immunostaining was decreased dramatically by postnatal day 21. Observations at the ultrastructural level confirmed a neuronal localization of TrkB and TrkC. Immunostaining for both receptors was restricted largely to the postsynaptic density of synaptic profiles in both dendrites and somata. In summary, this study illustrates a differential pattern of immunoreactivity between three neurotrophin receptors during development. The general increase of TrkB expression is well correlated with the onset of sound-evoked activity in this system, and its synaptic localization suggests that it may be involved in the modulation or maintenance of postsynaptic physiology.

Developmental influence of glycinergic transmission: regulation of NMDA receptor-mediated EPSPs.

The influence of excitatory transmission on postsynaptic structure is well established in developing animals, but little is known about the role of synaptic inhibition. We addressed this issue in developing gerbils with two manipulations designed to decrease glycinergic transmission in an auditory nucleus, the lateral superior olive (LSO), before the onset of sound-evoked activity. First, contralateral cochlear ablation functionally denervated the glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the LSO, while leaving the excitatory pathway intact. Second, continuous release of a glycine receptor antagonist, strychnine (SN), was used to decrease transmission. The strength of excitatory and inhibitory synapses was examined with whole-cell recordings from LSO neurons in a brain-slice preparation. The percentage of LSO neurons exhibiting MNTB-evoked IPSPs was reduced in both ablated and SN-treated animals. In those neurons displaying IPSPs, the amplitude was significantly reduced. This decrease was accompanied by an 8 mV depolarization in the IPSP equilibrium potential. In contrast, the ipsilaterally evoked EPSPs were of unusually long duration in experimental animals. These long-duration EPSPs were significantly shortened by hyperpolarizing the neuron to -90 mV or exposing them to aminophosphonopentanoic acid (AP-5), an NMDA receptor antagonist. Membrane hyperpolarization and AP-5 had little effect in control neurons. In addition, LSO neurons from ablated or SN-treated animals displayed broad rebound depolarizations after membrane hyperpolarization, and these were abolished in the presence of Ni2+. Because both cochlear ablation and SN-rearing were initiated before the onset of sound-evoked activity, the results suggest that spontaneous glycinergic transmission influences the development of postsynaptic properties, including the IPSP reversal potential, NMDA receptor function, and a Ca2+ conductance.