N-methyl-D-aspartate (NMDA) receptor subunit-specific probes were used to characterize developmental changes in the distribution of excitatory amino acid receptors in the chicken’s auditory brainstem nuclei. Although NR1 subunit expression does not change greatly during the development of the cochlear nuclei in the chicken (Tang and Carr  Hear. Res 191:79–89), there are significant developmental changes in NR2 subunit expression. We used in situ hybridization against NR1, NR2A, NR2B, NR2C, and NR2D to compare NR1 and NR2 expression during development. All five NMDA subunits were expressed in the auditory brainstem before embryonic day (E) 10, when electrical activity and synaptic responses appear in the nucleus magnocellularis (NM) and the nucleus laminaris (NL). At this time, the dominant form of the receptor appeared to contain NR1 and NR2B. NR2A appeared to replace NR2B by E14, a time that coincides with synaptic refinement and evoked auditory responses. NR2C did not change greatly during auditory development, whereas NR2D increased from E10 and remained at fairly high levels into adulthood. Thus changes in NMDA NR2 receptor subunits may contribute to the development of auditory brainstem responses in the chick.
cochlear nucleus; magnocellularis; laminaris; angularis; tonotopic gradient
The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the pan-KCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although pan-KCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally.
chicken; barn owl; ontogeny; time coding; outward current; high threshold
Caspase-3 is a cysteine protease that is most commonly associated with cell death. Recent studies have shown additional roles in mediating cell differentiation, cell proliferation and development of cell morphology. We investigated the role of caspase-3 in the development of chick auditory brainstem nuclei during embryogenesis. Immunofluorescence from embryonic days E6–13 revealed that the temporal expression of cleaved caspase-3 follows the ascending anatomical pathway. The expression is first seen in the auditory portion of VIIIth nerve including central axonal regions projecting to nucleus magnocellularis (NM), then later in NM axons projecting to nucleus laminaris (NL), and subsequently in NL dendrites. To examine the function of cleaved caspase-3 in chick auditory brainstem development, we blocked caspase-3 cleavage in developing chick embryos with the caspase-3 inhibitor Z-DEVD-FMK from E6 to E9, then examined NM and NL morphology and NM axonal targeting on E10. NL lamination in treated embryos was disorganized and the neuropil around NL contained a significant number of glial cells normally excluded from this region. Additionally, NM axons projected into inappropriate portions of NL in Z-DEVD-FMK treated embyros. We found that the presence of misrouted axons was associated with more severe NL disorganization. The effects of axonal caspase-3 inhibition on both NL morphogenesis and NM axon targeting suggest that these developmental processes are coordinated, likely through communication between axons and their targets.
caspase; auditory; brainstem; development; hindbrain; magnocellularis; laminaris; astrocytes
Identification of shared features between avian and mammalian auditory brainstem circuits has provided much insight into the mechanisms underlying early auditory processing. However, previous studies have highlighted an apparent difference in inhibitory systems; synaptic inhibition is thought to be slow and GABAergic in birds, but to have fast kinetics and be predominantly glycinergic in mammals. Using patch-clamp recordings in chick brainstem slices, we found this distinction is not exclusively true. Consistent with previous work, inhibitory postsynaptic currents (IPSCs) in nucleus magnocellularis (NM) were slow and mediated by GABAA receptors. However, IPSCs in nucleus laminaris (NL) and a subset of neurons in nucleus angularis (NA) had rapid time courses two to three-fold faster than those in NM. Further, we found IPSCs in NA were mediated by both glycine and GABAA receptors, demonstrating for the first time a role for fast glycinergic transmission in the avian auditory brainstem. Although NM, NL and NA have unique roles in auditory processing, the majority of inhibitory input to each nucleus arises from the same source, ipsilateral superior olivary nucleus (SON). Our results demonstrate remarkable diversity of inhibitory transmission among the avian brainstem nuclei and suggest differential glycine and GABAA receptor activity tailors inhibition to the specific functional roles of NM, NL, and NA despite common SON input. We additionally observed that glycinergic/GABAergic activity in NA was usually depolarizing and could elicit spiking activity in NA neurons. Because NA projects to SON, these excitatory effects may influence the recruitment of inhibitory activity in the brainstem nuclei.
Auditory; GABA; Glycine; Patch Clamp; Inhibition; Synapse
Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.
Millions of people suffer from debilitating hearing defects, ranging from a complete inability to detect sound to more subtle changes in how sounds are encoded by the nervous system. Many forms of deafness are due to mutations in genes that impair the development or function of hair cells, which are responsible for changing sound into electrical signals that can be processed by the brain. Both mice and humans carrying these mutations fail standard hearing tests. In contrast, very little is known about the genetic basis of central auditory processing disorders, which are poorly defined and difficult to diagnose, since these patients can still detect sounds. By finding genes that are required for the normal wiring of central auditory circuits in mice, we can investigate how changes at the circuit level affect circuit function and therefore improve our understanding of central auditory processing disorders. Here, we show that the natriuretic peptide receptor Npr2 is required to establish frequency maps in the mouse central auditory system. Surprisingly, despite a dramatic change in circuit organization, Npr2 mutant mice are still able to respond to sounds with normal sensitivity and timing, underscoring the need for better hearing diagnostic methods in mice as in humans.
For all neurons, a proper balance of synaptic excitation and inhibition is crucial to effect computational precision. Achievement of this balance is remarkable when one considers factors that modulate synaptic strength operate on multiple overlapping time scales and affect both pre- and postsynaptic elements. Recent studies have shown that inhibitory transmitters, glycine and GABA, are co-released in auditory nuclei involved in the computation of interaural time disparities (ITDs), a cue used to process sound source location. The co-release expressed at these synapses is heavily activity dependent, and generally occurs when input rates are high. This circuitry, in both birds and mammals, relies on inhibitory input to maintain the temporal precision necessary for ITD encoding. Studies of co-release in other brain regions suggest that GABA and glycine receptors (GlyRs) interact via cross-suppressive modulation of receptor conductance. We performed in vitro whole-cell recordings in several nuclei of the chicken brainstem auditory circuit to assess whether this cross-suppressive phenomenon was evident in the avian brainstem. We evaluated the effect of pressure-puff applied glycine on synaptically evoked inhibitory currents in nucleus magnocellularis (NM) and the superior olivary nucleus (SON). Glycine pre-application reduced the amplitude of inhibitory postsynaptic currents (IPSCs) evoked during a 100 Hz train stimulus in both nuclei. This apparent glycinergic modulation was blocked in the presence of strychnine. Further experiments showed that this modulation did not depend on postsynaptic biochemical interactions such as phosphatase activity, or direct interactions between GABA and GlyR proteins. Rather, voltage clamp experiments in which we manipulated Cl− flux during agonist application suggest that activation of one receptor will modulate the conductance of the other via local changes in Cl− ion concentration within microdomains of the postsynaptic membrane.
glycine; GABA; inhibition; cross-suppression; interaural time disparities
Age-related hearing loss – presbycusis – is the number one communication disorder and most prevalent neurodegenerative condition of our aged population. Although speech understanding in background noise is quite difficult for those with presbycusis, there are currently no biomedical treatments to prevent, delay or reverse this condition. A better understanding of the cochlear mechanisms underlying presbycusis will help lead to future treatments. Objectives of the present study were to investigate gamma-amino butyric acid A (GABAA) receptor subunit α1, nicotinic acetylcholine (nACh) receptor subunit β2, and N-methyl-D-aspartate (NMDA) receptor subunit NR1 mRNA and protein expression changes in spiral ganglion neurons of the CBA/CaJ mouse cochlea, that occur in age-related hearing loss, utilizing quantitative immunohistochemistry and semi-quantitative RT-PCR techniques. We found that auditory brainstem response (ABR) thresholds shifted over 40 dB from 3–48 kHz in old mice compared to young adults. DPOAE thresholds also shifted over 40 dB from 6–49 kHz in old mice, and their amplitudes were significantly decreased or absent in the same frequency range. Spiral ganglion neuron (SGN) density decreased with age in basal, middle and apical turns, and SGN density of the basal turn declined the most. A positive correlation was observed between SGN density and ABR wave 1 amplitude. mRNA and protein expression of GABAAR α1 and AChR β2 decreased with age in SGNs in the old mouse cochlea. mRNA and protein expression of NMDAR NR1 increased with age in SGNs of the old mice. These findings demonstrate that there are functionally-relevant age-related changes of GABAAR, nAChR, NMDAR expression in CBA mouse SGNs reflecting their degeneration, which may be related to functional changes in cochlear synaptic transmission with age, suggesting biological mechanisms for peripheral age-related hearing loss.
Aging; Hearing loss; Cochlea; Spiral Ganglion Neurons; Gene expression; Protein expression
NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-α to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
Long-lasting changes in communication between nerve cells require the regulation of gene expression. The influx of calcium ions into the cell, particularly through membrane protein called NMDA receptors, plays a crucial role in this process by determining the type of gene expression induced. NMDA receptors can exert multiple and very divergent effects within neuronal cells by impacting opposing phenomena such as synaptic plasticity and neuronal degeneration. We identified a protein termed Jacob that appears to play a pivotal role in such processes by entering the nucleus in response to NMDA receptor activation and controlling gene expression that governs cell survival and the stability of synaptic cell contacts. Removal of Jacob from the nucleus protects neurons from NMDA receptor–induced cell death and increases phosphorylation of the transcription factor CREB, whereas the opposite occurs after targeting Jacob exclusively to the nucleus. The work defines a novel pathway of synapse-to-nucleus communication involved in modelling synapto-dendritic input and NMDA receptor–induced cellular degeneration.
A new signaling mechanism from NMDA receptors to the nucleus plays an important role in the phosphorylation of the transcription factor CREB and neuronal cell survival.
The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)–P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease.
SIGNIFICANCE STATEMENT The subthalamic nucleus (STN) is a key component of the basal ganglia, a group of subcortical nuclei that control movement and are dysregulated in movement disorders such as Parkinson's disease. Subthalamic neurons receive direct excitatory input, but the pharmacology of excitatory synaptic transmission in the STN has been understudied. Here, we show that GluN2B- and GluN2D-containing NMDA receptors mediate the NMDA receptor component of EPSCs in subthalamic neurons. Moreover, our results demonstrate that pharmacologic modulation of GluN2D-containing receptors alters the time course of EPSCs and controls the in vivo spike-firing rate in the STN. This study identifies GluN2D as a potential target for modulating subthalamic neuron activity.
excitatory synapse; GluN2B; GluN2D; glutamate receptor; NMDA; subthalamic nucleus
Glutamatergic synaptic transmission in the mammalian central nervous system was slowly established over a period of some 20 years, dating from the 1950s. Realisation that glutamate and like amino acids (collectively known as excitatory amino acids (EAA)) mediated their excitatory actions via multiple receptors preceded establishment of these receptors as synaptic transmitter receptors. EAA receptors were initially classified as N-methyl-D-aspartate (NMDA) and non-NMDA receptors, the latter subdivided into quisqualate (later AMPA) and kainate receptors after agonists that appeared to activate these receptors preferentially, and by their sensitivity to a range of differentially acting antagonists developed progressively during the 1970s. NMDA receptors were definitively shown to be synaptic receptors on spinal neurones by the sensitivity of certain excitatory pathways in the spinal cord to a range of specific NMDA receptor antagonists. Importantly, specific NMDA receptor antagonists appeared to be less effective at synapses in higher centres. In contrast, antagonists that also blocked non-NMDA as well as NMDA receptors were almost universally effective at blocking synaptic excitation within the brain and spinal cord, establishing both the existence and ubiquity of non-NMDA synaptic receptor systems throughout the CNS. In the early 1980s, NMDA receptors were shown to be involved in several central synaptic pathways, acting in concert with non-NMDA receptors under conditions where a protracted excitatory postsynaptic potential was effected in response to intense stimulation of presynaptic fibres. Such activation of NMDA receptors together with non-NMDA receptors led to the phenomenon of long-term potentiation (LTP), associated with lasting changes in synaptic efficacy (synaptic plasticity) and considered to be an important process in memory and learning. During the 1980s, it was shown that certain glutamate receptors in the brain mediated biochemical changes that were not susceptible to NMDA or non-NMDA receptor antagonists. This dichotomy was resolved in the early 1990s by the techniques of molecular biology, which identified two families of glutamate-binding receptor proteins (ionotropic (iGlu) and metabotropic (mGlu) receptors). Development of antagonists binding to specific protein subunits is currently enabling precise identification of discrete iGlu or mGlu receptor subtypes that participate in a range of central synaptic processes, including synaptic plasticity.
L-Glutamate; excitatory amino acids; ionotropic glutamate receptors; metabotropic glutamate receptors; synaptic transmission
Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proven problematic. As a result, experiments aimed at genetic manipulations on birds remained difficult for this popular research tool. Recently, lentiviral methods have enabled production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal-specificity and stable expression of eGFP across generations (termed here as GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of gene manipulation, we compared the development, organization, structure and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) to that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM) and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.
transgenic quail; auditory brainstem
Defined as reduced neural responses during high rates of activity, synaptic depression is a form of short-term plasticity important for the temporal filtering of sound. In the avian cochlear nucleus magnocellularis (NM), an auditory brainstem structure, mechanisms regulating short-term synaptic depression include pre-, post-, and extrasynaptic factors. Using varied paired-pulse stimulus intervals, we found that the time course of synaptic depression lasts up to four seconds at late-developing NM synapses. Synaptic depression was largely reliant on exogenous Ca2+-dependent probability of presynaptic neurotransmitter release, and to a lesser extent, on the desensitization of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor (AMPA-R). Interestingly, although extrasynaptic glutamate clearance did not play a significant role in regulating synaptic depression, blocking glutamate clearance at early-developing synapses altered synaptic dynamics, changing responses from depression to facilitation. These results suggest a developmental shift in the relative reliance on pre-, post-, and extrasynaptic factors in regulating short-term synaptic plasticity in NM.
AMPA receptors; desensitization; development; glutamate transporters; nucleus magnocellularis; short-term synaptic depression
Nucleus laminaris (NL) neurons in the avian auditory brainstem are coincidence detectors necessary for the computation of interaural time differences used in sound localization. In addition to their excitatory inputs from nucleus magnocellularis, NL neurons receive inhibitory inputs from the superior olivary nucleus (SON) that greatly improve coincidence detection in mature animals. The mechanisms that establish mature distributions of inhibitory inputs to NL are not known. We used the vesicular GABA transporter (VGAT) as a marker for inhibitory presynaptic terminals to study the development of inhibitory inputs to NL between embryonic day 9 (E9) and E17. VGAT immunofluorescent puncta were first seen sparsely in NL at E9. The density of VGAT puncta increased with development, first within the ventral NL neuropil region and subsequently throughout both the ventral and dorsal dendritic neuropil, with significantly fewer terminals in the cell body region. A large increase in density occurred between E13–15 and E16–17, at a developmental stage when astrocytes that express glial fibrillary acidic protein (GFAP) become mature. We cultured E13 brainstem slices together with astrocyte-conditioned medium (ACM) obtained from E16 brainstems and found that ACM, but not control medium, increased the density of VGAT puncta. This increase was similar to that observed during normal development. Astrocyte-secreted factors interact with the terminal ends of SON axons to increase the number of GABAergic terminals. These data suggest that factors secreted from GFAP-positive astrocytes promote maturation of inhibitory pathways in the auditory brainstem.
astrocytes; inhibitory synapses; auditory brainstem; nucleus laminaris; synaptogenesis; superior olivary nucleus
The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits.
N-Methyl-d-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.
Information processing in the brain is carried out by networks of neurons connected by synapses. Synapses can change strength, allowing these networks to adapt and learn, in a process known as synaptic plasticity. At a synapse, an electrical signal in one neuron is converted into a chemical signal, carried by a neurotransmitter, which is in turn converted into electrical and chemical signals in another neuron by specialized proteins called receptors. One such protein, the N-methyl-d-aspartic acid (NMDA) receptor, is particularly important for plasticity, due to its ability to detect the voltage of the cell receiving the neurotransmitter signal and to the fact that it allows calcium, an important signaling molecule, to enter the cell. Here we use computational modeling to investigate the role of one part of the NMDA receptor: the NR2 subunit. The subunit has various forms, and which of these forms are present in the NMDA receptor can strongly affect the kinetics and other properties of the receptor. We show that, along with changing the kinetics of the receptor, changing the NR2 subunit affects the reliability of the receptor, its ability to respond to large stimuli, and its spatial response properties. These results have implications for synaptic transmission and plasticity.
Chronic tinnitus has no broadly effective treatment. Identification of specific markers for tinnitus should facilitate the development of effective therapeutics. Recently it was shown that glutamatergic blockade in the cerebellar paraflocculus, using an antagonist cocktail was successful in reducing chronic tinnitus. The present experiment examined the effect of selective N-methyl d-aspartate (NMDA) receptor blockade on tinnitus and associated spontaneous brain activity in a rat model. The NMDA antagonist, D(−)-2-amino-5-phosphonopentanoic acid (D-AP5) (0.5 mM), was continuously infused for 2 weeks directly to the ipsilateral paraflocculus of rats with tinnitus induced months prior by unilateral noise exposure. Treated rats were compared to untreated normal controls without tinnitus, and to untreated positive controls with tinnitus. D-AP5 significantly decreased tinnitus within three days of beginning treatment, and continued to significantly reduce tinnitus throughout the course of treatment and for 23 days thereafter, at which time testing was halted. At the conclusion of psychophysical testing, neural activity was assessed using manganese enhanced magnetic resonance imaging (MEMRI). In agreement with previous research, untreated animals with chronic tinnitus showed significantly elevated bilateral activity in their paraflocculus and brainstem cochlear nuclei, but not in mid or forebrain structures. In contrast, D-AP5-treated-tinnitus animals showed significantly less bilateral parafloccular and dorsal cochlear nucleus activity, as well as significantly less contralateral ventral cochlear nucleus activity. It was concluded that NMDA-mediated glutamatergic transmission in the paraflocculus appears to be a necessary component of chronic noise-induced tinnitus in a rat model. Additionally, it was confirmed that in this model, elevated spontaneous activity in the cerebellar paraflocculus and auditory brainstem is associated with tinnitus.
Hearing relies on Ca2+ influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca2+ channel subunit CaVβ2 in hearing. Of the CaVβ1–4 mRNAs, IHCs predominantly contained CaVβ2. Hearing was severely impaired in mice lacking CaVβ2 in extracardiac tissues (CaVβ2−/−). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in CaVβ2−/− mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. CaVβ2−/− IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing CaV1.3 channels. Confocal Ca2+ imaging revealed presynaptic Ca2+ microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer CaV1.3 channels. The coupling of the remaining Ca2+ influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the CaVβ2−/− mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in CaVβ2−/− mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic CaV1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca2+ activated potassium channels in IHCs were impaired in the absence of CaVβ2. Moreover, we found the developmental efferent innervation to persist in CaVβ2-deficient IHCs. In summary, CaVβ2 has an essential role in regulating the abundance and properties of CaV1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.
The chick embryo (Gallus domesticus) is one of the most important model systems in vertebrate developmental biology. The development and function of its auditory brainstem circuitry is exceptionally well studied. These circuits represent an excellent system for genetic manipulation to investigate mechanisms controlling neural circuit formation, synaptogenesis, neuronal polarity and dendritic arborization. The present study investigates the auditory nucleus, nucleus magnocellularis (NM). The neurotrophin receptor TrkB regulates dendritic structure in CNS neurons. TrkB is expressed at NM neurons at E7–E8 when these neurons have dendritic arbors. Downregulation of TrkB occurs after E8 followed by retraction of dendrites and by E18 most NM cells are adendritic. Is cessation of TrkB expression in NM necessary for dendritic retraction? To answer this question we combined focal in ovo electroporation with transposon mediated gene transfer to obtain stable expression of Doxycycline (Dox) regulated transgenes, specifically TrkB coexpressed with EGFP in a temporally controlled manner. Electroporation was performed at E2 and Dox added onto the chorioallointoic membrane from E7.5 to E16. Expression of EGFP had no effect on development of the embryo, or cell morphology and organization of auditory brainstem nuclei. NM cells expressing EGFP and TrkB at E17–E18 had dendrites and biophysical properties uncharacteristic for normal NM cells, indicating that cessation of TrkB expression is essential for dendrite retraction and functional maturation of these neurons. These studies indicate that expression of transposon based plasmids is an effective method to genetically manipulate events in mid to late embryonic brain development in chick.
Barn owls are capable of great accuracy in detecting the interaural time differences (ITDs) that underlie azimuthal sound localization. They compute ITDs in a circuit in nucleus laminaris (NL) that is reorganized with respect to birds like the chicken. The events that lead to the reorganization of the barn owl NL take place during embryonic development, shortly after the cochlear and laminaris nuclei have differentiated morphologically. At first the developing owl’s auditory brainstem exhibits morphology reminiscent of that of the developing chicken. Later, the two systems diverge, and the owl’s brainstem auditory nuclei undergo a secondary morphogenetic phase during which NL dendrites retract, the laminar organization is lost, and synapses are redistributed. These events lead to the restructuring of the ITD coding circuit and the consequent reorganization of the hindbrain map of ITDs and azimuthal space.
avian development; morphogenesis; auditory; laminaris; evolution; interaural time difference
Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.
Sound perception and sound localization are controlled by two distinct circuits in the central nervous system. However, the cellular and molecular determinants underlying their development are poorly understood. Here, we show that a spatially restricted region of the brainstem, the rhombomere 4, and two members of the Hox gene family, Hoxb1 and Hoxb2, are directly implicated in the development of the circuit leading to sound perception and sound amplification. In the absence of Hoxb1 and Hoxb2 function, we found severe morphological defects in the hair cell population implicated in transducing the acoustic signal, leading ultimately to severe hearing impairments in adult mutant mice. In contrast, the expression in the cochlear nucleus of another Hox member, Hoxa2, regulates the guidance receptor Rig1 and contralateral connectivity in the sound localization circuit. Some of the auditory dysfunctions described in our mouse models resemble pathological hearing conditions in humans, in which patients have an elevated hearing threshold sensitivity, as recorded in audiograms. Thus, this study provides mechanistic insight into the genetic and functional regulation of Hox genes during development and assembly of the auditory system.
N-Methyl-D-aspartate receptors (NMDA-Rs) are ion channels that are important for synaptic plasticity, which is involved in learning and drug addiction. We show enzymatic targeting of an NMDA-R antagonist, MK801, to a molecularly defined neuronal population with the cell-type-selectivity of genetic methods and the temporal control of pharmacology. We find that NMDA-Rs on dopamine neurons are necessary for cocaine-induced synaptic potentiation, demonstrating that cell type-specific pharmacology can be used to dissect signaling pathways within complex brain circuits.
Learning is critical to survival for humans and other animals. The learning process is regulated by receptors on the surface of brain cells called N-Methyl-D-aspartate receptors (or NMDA receptors for short). These receptors help to strengthen signals between brain cells, which allows a new concept or action to be learned. However, it has been difficult to pin down how the role of NMDA receptors selectively in specific types of brain cells. While drugs can be used to quickly block NMDA receptors throughout the brain, it is hard to target drugs to a specific cell type. Also, genetic engineering can be used to selectively knock out NMDA receptors in certain types of brain cells, but these techniques are too slow, and can take weeks or even a lifetime to work.
Now, Yang et al. have developed a clever way to combine an NMDA-blocking drug and genetic engineering to study NMDA receptors' responses to cocaine in specific brain cells. This approach involved first creating an inactive form of an NMDA-blocking drug that can only becomes active when it is processed by an enzyme that is normally produced in pigs' livers. Next, living mouse brain cells, including some that were engineered to express the pig enzyme, were exposed to the drug in the laboratory. The drug blocked the NMDA receptors on brain cells that expressed the enzyme, but not the receptors on nearby brain cells that lacked the enzyme. This occurred even though all the cells produced NMDA receptors and all were exposed to the drug.
NMDA receptors have been known to play an important role in cocaine addiction for more than 20 years. Drugs like cocaine can co-opt the normally healthy learning process involving NMDA receptors and lead to a maladaptive form of learning that is commonly called addiction. Cocaine strengthens signals between brain cells causing the behaviors associated with using cocaine to become deeply ingrained and difficult to change. Yang et al. used cell type-specific targeting of a drug that blocks NMDA receptors to observe what happened in cocaine-exposed brain cells with, or without, working NMDA receptors.
As expected, the experiments showed that cocaine didn't strengthen brain signals in cells without working NMDA receptors. Specifically, the experiments showed that NMDA receptors on a type of brain cell that release a pleasure-inducing chemical called dopamine are necessary for cocaine–induced synaptic plasticity. The combination technique developed by Yang et al. will likely be used by other scientists to further study the role of NMDA receptors in specific brain cells during addiction and normal brain activity.
NMDA receptor; cell type-specific pharmacology; dopamine; cocaine; synaptic plasticity; mouse
Auditory information is important for social and reproductive behaviors in birds generally, but is crucial for oscine species (songbirds), in particular because in these species auditory feedback ensures the learning and accurate maintenance of song. While there is considerable information on the auditory projections through the forebrain of songbirds, there is no information available for projections through the brainstem. At the latter levels the prevalent model of auditory processing in birds derives from an auditory specialist, the barn owl, which uses time and intensity parameters to compute the location of sounds in space, but whether the auditory brainstem of songbirds is similarly functionally organized is unknown. To examine the songbird auditory brainstem we charted the projections of the cochlear nuclei angularis (NA) and magnocellularis (NM) and the third-order nucleus laminaris (NL) in zebra finches using standard tract-tracing techniques. As in other avian species, the projections of NM were found to be confined to NL, and NL and NA provided the ascending projections. Here we report on differential projections of NA and NL to the torus semicircularis, known in birds as nucleus mesencephalicus lateralis, pars dorsalis (MLd), and in mammals as the central nucleus of the inferior colliculus (ICc). Unlike the case in nonsongbirds, the projections of NA and NL to MLd in the zebra finch showed substantial overlap, in agreement with the projections of the cochlear nuclei to the ICc in mammals. This organization could suggest that the “what” of auditory stimuli is as important as “where.”
cochlear nuclei; central nucleus of inferior colliculus; MLd; zebra finch; avian
Topographic organization of neurons is a hallmark of brain structure. The establishment of the connections between topographically organized brain regions has attracted much experimental attention and it is widely accepted that molecular cues guide outgrowing axons to their targets in order to construct topographic maps. In a number of systems afferent axons are organized topographically along their trajectory as well and it has been suggested that this pre-target sorting contributes to map formation. Neurons in auditory regions of the brain are arranged according to their best frequency (BF), the sound frequency they respond to optimally. This BF changes predictably with position along the so-called tonotopic axis. In the avian auditory brainstem, the tonotopic organization of the second- and third-order auditory neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL) has been well described. In this study we examine whether the decussating NM axons forming the crossed dorsal cochlear tract (XDCT) and innervating the contralateral NL are arranged in a systematic manner. We electroporated dye into cells in different frequency regions of NM to anterogradely label their axons in the XDCT. The placement of dye in NM was compared to the location of labeled axons in XDCT. Our results show that NM axons in XDCT are organized in a precise tonotopic manner along the rostrocaudal axis, spanning over the entire rostrocaudal extent of both the origin and target nuclei. We propose that in the avian auditory brainstem, this pre-target axon sorting contributes to tonotopic map formation in NL.
axon topography; pre-target axon sorting; auditory system; tonotopic organization; sound localization
The basic helix-loop-helix (bHLH) transcription factor Math5 (Atoh7) is required for retinal ganglion cell (RGC) and optic nerve development. Using Math5-lacZ knockout mice, we have identified an additional expression domain for Math5 outside the eye, in functionally connected structures of the central auditory system. In the adult hindbrain, the cytoplasmic Math5-lacZ reporter is expressed within the ventral cochlear nucleus (VCN), in a subpopulation of neurons that project to medial nucleus of the trapezoid body (MNTB), lateral superior olive (LSO), and lateral lemniscus (LL). These cells were identified as globular and small spherical bushy cells based on their morphology, abundance, distribution within the cochlear nucleus (CN), co-expression of Kv1.1, Kv3.1b and Kcnq4 potassium channels, and projection patterns within the auditory brainstem. Math5-lacZ is also expressed by cochlear root neurons in the auditory nerve. During embryonic development, Math5-lacZ was detected in precursor cells emerging from the caudal rhombic lip from embryonic day (E)12 onwards, consistent with the time course of CN neurogenesis. These cells co-express MafB, Math1 and Math5 and are post-mitotic. Math5 expression in the CN was verified by mRNA in situ hybridization, and the identity of positive neurons was confirmed morphologically using a Math5-Cre BAC transgene with an alkaline phosphatase reporter.
The hindbrains of Math5 mutants appear grossly normal, with the exception of the CN. Although overall CN dimensions are unchanged, the lacZ positive cells are significantly smaller in Math5 −/− mice compared to Math5 +/− mice, suggesting these neurons may function abnormally. The Auditory Brainstem Response (ABR) of Math5 mutants was evaluated in a BALB/cJ congenic background. ABR thresholds of Math5 −/− mice were similar to those of wild-type and heterozygous mice, but the interpeak latencies for Peaks II-IV were significantly altered. These temporal changes are consistent with a higher level auditory processing disorder involving the CN, potentially affecting the integration of binaural sensory information.
Math5; cochlear nucleus; AVCN; PVCN; mouse; atonal; Math1; bHLH; globular bushy cells; spherical bushy cells; cochlear root neurons; auditory processing; ABR latency; binaural integration; interaural intensity difference; interaural time difference; phase locking; Calyx of Held; Kv1.1; Kv3.1; Kcnq4; mafB; Cre recombinase; fluorogold; tract-tracing; inferior colliculus; multipolar cells; rhombic lip
Neurons restore their function in response to external or internal perturbations and maintain neuronal or network stability through a homeostatic scaling mechanism. Homeostatic responses at synapses along the auditory system would be important for adaptation to normal and abnormal fluctuations in the sensory environment. We investigated at the electron microscopic level and after postembedding immunogold labeling whether projection neurons in the cochlear nucleus responded to modifications of auditory nerve activity. After unilaterally reducing the level of auditory inputs by ~ 20 dB by monaural earplugging, auditory nerve synapses on bushy cells somata and basal dendrites of fusiform cells of the ventral and dorsal cochlear nucleus, respectively, upregulated GluR3 AMPA receptor subunit, while inhibitory synapses decreased the expression of GlyRα1 subunit. These changes in expression levels were fully reversible once the earplug was removed, indicating that activity affects the trafficking of receptors at synapses. Excitatory synapses on apical dendrites of fusiform cells (parallel fibers) with different synaptic AMPA receptor subunit composition, were not affected by sound attenuation, as the expression levels of AMPA receptor subunits were the same as in normal hearing littermates. GlyRα1 subunit expression at inhibitory synapses on apical dendrites of fusiform cells was also found unaffected. Furthermore, fusiform and bushy cells of the contralateral side to the earplugging upregulated the GluR3 subunit at auditory nerve synapses. These results show that cochlear nucleus neurons innervated by the auditory nerve, are able to respond to small changes in sound levels by redistributing specific AMPA and glycine receptor subunits.
earplugging; glutamate receptors; glycine α1 subunit; postembedding immunogold labeling; ultrastructure