In contrast to our knowledge about the anatomical development of the mammalian central auditory system, the development of its physiological properties is still poorly understood. In order to better understand the physiological properties of the developing mammalian auditory brainstem, we made intracellular recordings in brainstem slices from perinatal rats to examine synaptic transmission in the superior olivary complex, the first binaural station in the ascending auditory pathway. We concentrated on neurons in the lateral superior olive (LSO), which in adults, are excited from the ipsilateral side and inhibited from the contralateral side. Already at embryonic day (E) 18, when axon collaterals begin to invade the LSO anlage, synaptic potentials could be evoked from ipsilateral, as well as from contralateral inputs. Ipsilaterally elicited PSPs were always depolarizing, regardless of age. They had a positive reversal potential and could be completely blocked by the non-NMDA glutamate receptor antagonist CNQX. In contrast, contralaterally elicited PSPs were depolarizing from E18-P4, yet they turned into "adult-like," hyperpolarizing PSPs after P8. Their reversal potential shifted dramatically from -21.6 +/- 17.7 mV (E18-P0) to -73.0 +/- 7.1 mV (P10). Regardless of their polarity, contralaterally elicited PSPs were reversibly blocked by the glycine receptor antagonist strychnine. Bath application of glycine and its agonist beta-alanine further confirmed the transitory depolarizing action of glycine in the auditory brainstem. Since the transient excitatory behavior of glycine occurs during a period during which glycinergic synaptic connections in the LSO are refined by activity-dependent mechanisms, glycinergic excitation might be a mechanism by which synaptic rearrangement in the contralateral inhibitory pathway is accomplished.
Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration.
Western blot analysis is routinely employed for quantifying differences in protein levels between samples. To control equal loading and to arithmetically compensate loading differences, immunodetection of housekeeping proteins is commonly used. Due to potential biases, this approach has been criticized. Here, we evaluate epicocconone‐based total protein staining (E‐ToPS) as an alternative. We compared it with two other total protein stainings (Coomassie and Sypro Ruby) and with immunodetection of housekeeping proteins (β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase). Evaluation comprised both the natural and the synthetic epicocconone compound. Both compounds produced highly congruent results and showed more sensitive (≤ 1 μg) and less variable staining properties than the other variants. The high sensitivity of E‐ToPS, covering minute protein amounts, makes it a powerful loading control, especially for precious samples. Regarding biological and technical variances, E‐ToPS outperformed immunostaining against β‐tubulin and glyceraldehyde 3‐phosphate dehydrogenase. Furthermore, E‐ToPS had no impact on subsequent immunodetection, allowing for an early control of proper loading prior to immunodetection. In contrast to earlier studies, we found logarithmic staining properties for E‐ToPS, which should be considered when using it for arithmetic normalization. In conclusion, we demonstrate the superior power of E‐ToPS as a loading control for Western blots.
1. The development of excitatory activation in the visual cortex was studied in fetal and neonatal cats. During fetal and neonatal life, the immature cerebral cortex (the cortical plate) is sandwiched between two synaptic zones: the marginal zone above, and an area just below the cortical plate, the subplate. The subplate is transient and disappears by approximately 2 mo postnatal. Here we have investigated whether the subplate and the cortical plate receive functional synaptic inputs in the fetus, and when the adultlike pattern of excitatory synaptic input to the cortical plate appears during development. 2. Extracellular field potential recording to electrical stimulation of the optic radiation was performed in slices of cerebral cortex maintained in vitro. Laminar profiles of field potentials were converted by the current-source density (CSD) method to identify the spatial and temporal distribution of neuronal excitation within the subplate and the cortical plate. 3. Between embryonic day 47 (E47) and postnatal day 28 (P28; birth, E65), age-related changes occur in the pattern of synaptic activation of neurons in the cortical plate and the subplate. Early in development, at E47, E57, and P0, short-latency (probably monosynaptic) excitation is most obvious in the subplate, and longer latency (presumably polysynaptic) excitation can be seen in the cortical plate. Synaptic excitation in the subplate is no longer apparent at P21 and P28, a time when cell migration is finally complete and the cortical layers have formed. By contrast, excitation in the cortical plate is prominent in postnatal animals, and the temporal and spatial pattern has changed. 4. The adultlike sequence of synaptic activation in the different cortical layers can be seen by P28. It differs from earlier ages in several respects. First, short-latency (probably monosynaptic) excitation can be detected in cortical layer 4. Second, multisynaptic, long-lasting activation is present in layers 2/3 and 5. 5. Our results show that the subplate zone, known from anatomic studies to be a synaptic neurophil during development, receives functional excitatory inputs from axons that course in the developing white matter. Because the only mature neurons present in this zone are the subplate neurons, we conclude that subplate neurons are the principal, if not the exclusive, recipients of this input. The results suggest further that the excitation in the subplate in turn is relayed to neurons of the cortical plate via axon collaterals of subplate neurons.(ABSTRACT TRUNCATED AT 400 WORDS)
The voltage-activated L-type calcium channels Cav1.2 and Cav1.3 mediate Ca2+ influx into neurons at the soma or at dendrites, whereas they are not observed at the presynapse. Surprisingly, in the inner ear, Cav1.3 is indispensable for signal transmission from the presynaptic cochlear inner hair cells to the postsynaptic auditory nerve fibers. Due to Cav1.3 channel clustering at ribbons, i.e., specific presynaptic structures of the hair cells, they promote Ca2+ influx, which triggers calcium-dependent fusion of synaptic vesicles with the plasma membrane. Mutations in Cacna1d, a gene that encodes Cav1.3, result in deafness because release of the neurotransmitter glutamate at the synapses is abolished. Moreover, studies of the auditory pathway have revealed that Cav1.3 plays an important part in the central auditory system as well. Absence of the channel results in severe changes in auditory pathway cytoarchitecture and in abnormal electrophysiological performance of auditory neurons. Furthermore, developmental refinement of tonotopic inhibitory projections in sound localization circuits is disrupted. These aberrations are associated with abnormal sound processing in the auditory pathway. This goes to show that the Cav1.3 channel is essential for inner ear functioning as well as auditory pathway development and performance. Cacna1d therefore represents a prototypal deafness-associated gene, in which mutations result in both peripheral and central auditory deficiencies. This, in turn, has implications for auditory rehabilitation using cochlear implants that address only peripheral dysfunctions. Exploratory research into the closely related Cav1.2 isoform points to an important role of this channel in acoustic trauma. Cav1.2 is mainly expressed in the auditory nerve, but apparently not essential for normal auditory function. Loss of function of the channel, however, does influence the effects of traumatic noise exposure. Loss of this channel induced by noise trauma results in reduced auditory threshold increase—as compared with the control group. This phenomenon points to the fact that Cav1.2-mediated Ca2+ influx is involved in noise trauma-induced damage. Deeper insight into this function might result in new therapeutic approaches.
