GABAergic inhibition gates excitatory LTP in perirhinal cortex.

The perirhinal cortex (PRh) is a key region downstream of auditory cortex (ACx) that processes familiarity linked mnemonic signaling. In gerbils, ACx-driven EPSPs recorded in PRh neurons are largely shunted by GABAergic inhibition (Kotak et al., 2015, Frontiers in Neural Circuits, 9). To determine whether inhibitory shunting prevents the induction of excitatory long-term potentiation (e-LTP), we stimulated ACx-recipient PRh in a brain slice preparation using theta burst stimulation (TBS). Under control conditions, without GABA blockers, the majority of PRh neurons exhibited long-term depression. A very low concentration of bicuculline increased EPSP amplitude, but under this condition TBS did not significantly increase e-LTP induction. Since PRh synaptic inhibition included a GABAB receptor-mediated component, we added a GABAB receptor antagonist. When both GABAA and GABAB receptors were blocked, TBS reliably induced e-LTP in a majority of PRh neurons. We conclude that GABAergic transmission is a vital mechanism regulating e-LTP induction in the PRh, and may be associated with auditory learning.

Cortical Synaptic Inhibition Declines during Auditory Learning.

Auditory learning is associated with an enhanced representation of acoustic cues in primary auditory cortex, and modulation of inhibitory strength is causally involved in learning. If this inhibitory plasticity is associated with task learning and improvement, its expression should emerge and persist until task proficiency is achieved. We tested this idea by measuring changes to cortical inhibitory synaptic transmission as adult gerbils progressed through the process of associative learning and perceptual improvement. Using either of two procedures, aversive or appetitive conditioning, animals were trained to detect amplitude-modulated noise and then tested daily. Following each training session, a thalamocortical brain slice was generated, and inhibitory synaptic properties were recorded from layer 2/3 pyramidal neurons. Initial associative learning was accompanied by a profound reduction in the amplitude of spontaneous IPSCs (sIPSCs). However, sIPSC amplitude returned to control levels when animals reached asymptotic behavioral performance. In contrast, paired-pulse ratios decreased in trained animals as well as in control animals that experienced unpaired conditioned and unconditioned stimuli. This latter observation suggests that inhibitory release properties are modified during behavioral conditioning, even when an association between the sound and reinforcement cannot occur. These results suggest that associative learning is accompanied by a reduction of postsynaptic inhibitory strength that persists for several days during learning and perceptual improvement.

Transient Hearing Loss Within a Critical Period Causes Persistent Changes to Cellular Properties in Adult Auditory Cortex.

Sensory deprivation can induce profound changes to central processing during developmental critical periods (CPs), and the recovery of normal function is maximal if the sensory input is restored during these epochs. Therefore, we asked whether mild and transient hearing loss (HL) during discrete CPs could induce changes to cortical cellular physiology. Electrical and inhibitory synaptic properties were obtained from auditory cortex pyramidal neurons using whole-cell recordings after bilateral earplug insertion or following earplug removal. Varying the age of HL onset revealed brief CPs of vulnerability for membrane and firing properties, as well as, inhibitory synaptic currents. These CPs closed 1 week after ear canal opening on postnatal day (P) 18. To examine whether the cellular properties could recover from HL, earplugs were removed prior to (P17) or after (P23), the closure of these CPs. The earlier age of hearing restoration led to greater recovery of cellular function, but firing rate remained disrupted. When earplugs were removed after the closure of these CPs, several changes persisted into adulthood. Therefore, long-lasting cellular deficits that emerge from transient deprivation during a CP may contribute to delayed acquisition of auditory skills in children who experience temporary HL.

On the localization of complex sounds: temporal encoding based on input-slope coincidence detection of envelopes.

Behavioral and neural findings demonstrate that animals can locate low-frequency sounds along the azimuth by detecting microsecond interaural time differences (ITDs). Information about ITDs is also available in the amplitude modulations (i.e., envelope) of high-frequency sounds. Since medial superior olivary (MSO) neurons encode low-frequency ITDs, we asked whether they employ a similar mechanism to process envelope ITDs with high-frequency carriers, and the effectiveness of this mechanism compared with the process of low-frequency sound. We developed a novel hybrid in vitro dynamic-clamp approach, which enabled us to mimic synaptic input to brain-slice neurons in response to virtual sound and to create conditions that cannot be achieved naturally but are useful for testing our hypotheses. For each simulated ear, a virtual sound, computer generated, was used as input to a computational auditory-nerve model. Model spike times were converted into synaptic input for MSO neurons, and ITD tuning curves were derived for several virtual-sound conditions: low-frequency pure tones, high-frequency tones modulated with two types of envelope, and speech sequences. Computational models were used to verify the physiological findings and explain the biophysical mechanism underlying the observed ITD coding. Both recordings and simulations indicate that MSO neurons are sensitive to ITDs carried by spectrotemporally complex virtual sounds, including speech tokens. Our findings strongly suggest that MSO neurons can encode ITDs across a broad-frequency spectrum using an input-slope-based coincidence-detection mechanism. Our data also provide an explanation at the cellular level for human localization performance involving high-frequency sound described by previous investigators.

Hearing loss differentially affects thalamic drive to two cortical interneuron subtypes.

Sensory deprivation, such as developmental hearing loss, leads to an adjustment of synaptic and membrane properties throughout the central nervous system. These changes are thought to compensate for diminished sound-evoked activity. This model predicts that compensatory changes should be synergistic with one another along each functional pathway. To test this idea, we examined the excitatory thalamic drive to two types of cortical inhibitory interneurons that display differential effects in response to developmental hearing loss. The inhibitory synapses made by fast-spiking (FS) cells are weakened by hearing loss, whereas those made by low threshold-spiking (LTS) cells remain strong but display greater short-term depression (Takesian et al. 2010). Whole-cell recordings were made from FS or LTS interneurons in a thalamocortical brain slice, and medial geniculate (MG)-evoked postsynaptic potentials were analyzed. Following hearing loss, MG-evoked net excitatory potentials were smaller than normal at FS cells but larger than normal at LTS cells. Furthermore, MG-evoked excitatory potentials displayed less short-term depression at FS cells and greater short-term depression at LTS cells. Thus deprivation-induced adjustments of excitatory synapses onto inhibitory interneurons are cell-type specific and parallel the changes made by the inhibitory afferents.

Asymmetric excitatory synaptic dynamics underlie interaural time difference processing in the auditory system.

Low-frequency sound localization depends on the neural computation of interaural time differences (ITD) and relies on neurons in the auditory brain stem that integrate synaptic inputs delivered by the ipsi- and contralateral auditory pathways that start at the two ears. The first auditory neurons that respond selectively to ITD are found in the medial superior olivary nucleus (MSO). We identified a new mechanism for ITD coding using a brain slice preparation that preserves the binaural inputs to the MSO. There was an internal latency difference for the two excitatory pathways that would, if left uncompensated, position the ITD response function too far outside the physiological range to be useful for estimating ITD. We demonstrate, and support using a biophysically based computational model, that a bilateral asymmetry in excitatory post-synaptic potential (EPSP) slopes provides a robust compensatory delay mechanism due to differential activation of low threshold potassium conductance on these inputs and permits MSO neurons to encode physiological ITDs. We suggest, more generally, that the dependence of spike probability on rate of depolarization, as in these auditory neurons, provides a mechanism for temporal order discrimination between EPSPs.

Age-dependent effect of hearing loss on cortical inhibitory synapse function.

The developmental plasticity of excitatory synapses is well established, particularly as a function of age. If similar principles apply to inhibitory synapses, then we would expect manipulations during juvenile development to produce a greater effect and experience-dependent changes to persist into adulthood. In this study, we first characterized the maturation of cortical inhibitory synapse function from just before the onset of hearing through adulthood. We then examined the long-term effects of developmental conductive hearing loss (CHL). Whole cell recordings from gerbil thalamocortical brain slices revealed a significant decrease in the decay time of inhibitory currents during the first 3 mo of normal development. When assessed in adults, developmental CHL led to an enduring decrease of inhibitory synaptic strength, whereas the maturation of synaptic decay time was only delayed. Early CHL also depressed the maximum discharge rate of fast-spiking, but not low-threshold-spiking, inhibitory interneurons. We then asked whether adult onset CHL had a similar effect, but neither inhibitory current amplitude nor decay time was altered. Thus inhibitory synapse function displays a protracted development during which deficits can be induced by juvenile, but not adult, hearing loss. These long-lasting changes to inhibitory function may contribute to the auditory processing deficits associated with early hearing loss.

Normal hearing is required for the emergence of long-lasting inhibitory potentiation in cortex.

Long-term synaptic plasticity is a putative mechanism for learning in adults. However, there is little understanding of how synaptic plasticity mechanisms develop or whether their maturation depends on experience. Since inhibitory synapses are particularly malleable to sensory stimulation, long-lasting potentiation of inhibitory synapses was characterized in auditory thalamocortical slices. Intracortical high-frequency electrical stimulation led to a 67% increase in inhibitory synaptic currents. In the absence of stimulation, inhibitory potentiation was induced by a brief exposure to exogenous brain-derived neurotrophic factor (BDNF). BDNF exposure occluded any additional potentiation by high-frequency afferent stimulation, suggesting that BDNF signaling is sufficient to account for inhibitory potentiation. Moreover, inhibitory potentiation was reduced significantly by extracellular application of a BDNF scavenger or by intracellular blockade of BDNF receptor [tropomyosin-related kinase B (TrkB)] signaling. In contrast, glutamatergic or GABAergic antagonists did not prevent the induction of inhibitory potentiation. Since BDNF and TrkB expression are influenced strongly by activity, we predicted that inhibitory potentiation would be diminished by manipulations that decrease central auditory activity, such as hearing loss. Two forms of hearing loss were examined: conductive hearing loss in which the cochleae are not damaged or sensorineural hearing loss in which both cochleae are removed. Both forms of hearing loss were found to reduce significantly the magnitude of inhibitory potentiation. These data indicate that early experience is necessary for the normal development of BDNF-mediated long-lasting inhibitory potentiation, which may be associated with perceptual deficits at later ages.

Presynaptic GABA(B) receptors regulate experience-dependent development of inhibitory short-term plasticity.

Short-term changes in synaptic gain support information processing throughout the CNS, yet we know little about the developmental regulation of such plasticity. Here we report that auditory experience is necessary for the normal maturation of synaptic inhibitory short-term plasticity (iSTP) in the auditory cortex, and that presynaptic GABA(B) receptors regulate this development. Moderate or severe hearing loss was induced in gerbils, and iSTP was characterized by measuring inhibitory synaptic current amplitudes in response to repetitive stimuli. We reveal a profound developmental shift of iSTP from depressing to facilitating after the onset of hearing. Even moderate hearing loss prevented this shift. This iSTP change was mediated by a specific class of inhibitory interneurons, the low-threshold spiking cells. Further, using paired recordings, we reveal that presynaptic GABA(B) receptors at interneuron-pyramidal connections regulate iSTP in an experience-dependent manner. This novel synaptic mechanism may support the emergence of mature temporal processing in the auditory cortex.

Hearing loss prevents the maturation of GABAergic transmission in the auditory cortex.

Inhibitory neurotransmission is a critical determinant of neuronal network gain and dynamic range, suggesting that network properties are shaped by activity during development. A previous study demonstrated that sensorineural hearing loss (SNHL) in gerbils leads to smaller inhibitory potentials in L2/3 pyramidal neurons in the thalamorecipient auditory cortex, ACx. Here, we explored the mechanisms that account for proper maturation of gamma-amino butyric acid (GABA)ergic transmission. SNHL was induced at postnatal day (P) 10, and whole-cell voltage-clamp recordings were obtained from layer 2/3 pyramidal neurons in thalamocortical slices at P16-19. SNHL led to an increase in the frequency of GABAzine-sensitive (antagonist) spontaneous (s) and miniature (m) inhibitory postsynaptic currents (IPSCs), accompanied by diminished amplitudes and longer durations. Consistent with this, the amplitudes of minimum-evoked IPSCs were also reduced while their durations were longer. The alpha1- and beta2/3 subunit-specific agonists zolpidem and loreclezole increased control but not SNHL sIPSC durations. To test whether SNHL affected the maturation of GABAergic transmission, sIPSCs were recorded at P10. These sIPSCs resembled the long SNHL sIPSCs. Furthermore, zolpidem and loreclezole were ineffective in increasing their durations. Together, these data strongly suggest that the presynaptic release properties and expression of key postsynaptic GABA(A) receptor subunits are coregulated by hearing.

Hearing loss alters the subcellular distribution of presynaptic GAD and postsynaptic GABAA receptors in the auditory cortex.

We have shown previously that auditory experience regulates the maturation of excitatory synapses in the auditory cortex (ACx). In this study, we used electron microscopic immunocytochemistry to determine whether the heightened excitability of the ACx following neonatal sensorineural hearing loss (SNHL) also involves pre- or postsynaptic alterations of GABAergic synapses. SNHL was induced in gerbils just prior to the onset of hearing (postnatal day 10). At P17, the gamma-aminobutyri acid type A (GABA(A)) receptor's beta2/3-subunit (GABA(A)beta2/3) clusters residing at plasma membranes in layers 2/3 of ACx was reduced significantly in size (P < 0.05) and number (P < 0.005), whereas the overall number of immunoreactive puncta (intracellular + plasmalemmal) remained unchanged. The reduction of GABA(A)beta2/3 was observed along perikaryal plasma membranes of excitatory neurons but not of GABAergic interneurons. This cell-specific change can contribute to the enhanced excitability of SNHL ACx. Presynaptically, GABAergic axon terminals were significantly larger but less numerous and contained 47% greater density of glutamic acid decarboxylase immunoreactivity (P < 0.05). This suggests that GABA synthesis may be upregulated by a retrograde signal arising from lowered levels of postsynaptic GABA(A)R. Thus, both, the pre- and postsynaptic sides of inhibitory synapses that form upon pyramidal neurons of the ACx are regulated by neonatal auditory experience.

Conductive hearing loss disrupts synaptic and spike adaptation in developing auditory cortex.

Although sensorineural hearing loss (SNHL) is known to compromise central auditory structure and function, the impact of milder forms of hearing loss on cellular neurophysiology remains mostly undefined. We induced conductive hearing loss (CHL) in developing gerbils, reared the animals for 8-13 d, and subsequently assessed the temporal features of auditory cortex layer 2/3 pyramidal neurons in a thalamocortical brain slice preparation with whole-cell recordings. Repetitive stimulation of the ventral medial geniculate nucleus (MGv) evoked robust short-term depression of the postsynaptic potentials in control neurons, and this depression increased monotonically at higher stimulation frequencies. In contrast, CHL neurons displayed a faster rate of synaptic depression and a smaller asymptotic amplitude. Moreover, the latency of MGv evoked potentials was consistently longer in CHL neurons for all stimulus rates. A separate assessment of spike frequency adaptation in response to trains of injected current pulses revealed that CHL neurons displayed less adaptation compared with controls, although there was an increase in temporal jitter. For each of these properties, nearly identical findings were observed for SNHL neurons. Together, these data show that CHL significantly alters the temporal properties of auditory cortex synapses and spikes, and this may contribute to processing deficits that attend mild to moderate hearing loss.

The Sensory Striatum Is Permanently Impaired by Transient Developmental Deprivation.

Corticostriatal circuits play a fundamental role in regulating many behaviors, and their dysfunction is associated with many neurological disorders. In contrast, sensory disorders, like hearing loss (HL), are commonly linked with processing deficits at or below the level of the auditory cortex (ACx). However, HL can be accompanied by non-sensory deficits, such as learning delays, suggesting the involvement of regions downstream of ACx. Here, we show that transient developmental HL differentially affected the ACx and its downstream target, the sensory striatum. Following HL, both juvenile ACx layer 5 and striatal neurons displayed an excitatory-inhibitory imbalance and lower firing rates. After hearing was restored, adult ACx neurons recovered balanced excitatory-inhibitory synaptic gain and control-like firing rates, but striatal neuron synapses and firing properties did not recover. Thus, a brief period of abnormal cortical activity may induce cellular impairments that persist into adulthood and contribute to neurological disorders that are striatal in origin.

Hearing loss raises excitability in the auditory cortex.

Developmental hearing impairments compromise sound discrimination, speech acquisition, and cognitive function; however, the adjustments of functional properties in the primary auditory cortex (A1) remain unknown. We induced sensorineural hearing loss (SNHL) in developing gerbils and then reared the animals for several days. The intrinsic membrane and synaptic properties of layer 2/3 pyramidal neurons were subsequently examined in a thalamocortical brain slice preparation with whole-cell recordings and electron microscopic immunocytochemistry. SNHL neurons displayed a depolarized resting membrane potential, an increased input resistance, and a higher incidence of sustained firing. They also exhibited significantly larger thalamocortically and intracortically evoked excitatory synaptic responses, including a greater susceptibility to the NMDA receptor antagonist AP-5 and the NR2B subunit antagonist ifenprodil. This correlated with an increase in NR2B labeling of asymmetric synapses, as visualized ultrastructurally. Furthermore, decreased frequency and increased amplitude of miniature EPSCs (mEPSCs) in SNHL neurons suggest that a decline in presynaptic release properties is compensated by an increased excitatory response. To verify that the increased thalamocortical excitation was elicited by putative monosynaptic connections, minimum amplitude ventral medial geniculate nucleus-evoked EPSCs were recorded. These minimum-evoked responses were of larger amplitude, and the NMDAergic currents were also larger and longer in SNHL neurons. These findings were supported by significantly longer AP-5-sensitive durations and larger amplitudes of mEPSCs. Last, the amplitudes of intracortically evoked monosynaptic and polysynaptic GABAergic inhibitory synaptic responses were significantly smaller in SNHL neurons. These alterations in cellular properties after deafness reflect an attempt by A1 to sustain an operative level of cortical excitability that may involve homeostatic mechanisms.

The onset of visual experience gates auditory cortex critical periods.

Sensory systems influence one another during development and deprivation can lead to cross-modal plasticity. As auditory function begins before vision, we investigate the effect of manipulating visual experience during auditory cortex critical periods (CPs) by assessing the influence of early, normal and delayed eyelid opening on hearing loss-induced changes to membrane and inhibitory synaptic properties. Early eyelid opening closes the auditory cortex CPs precociously and dark rearing prevents this effect. In contrast, delayed eyelid opening extends the auditory cortex CPs by several additional days. The CP for recovery from hearing loss is also closed prematurely by early eyelid opening and extended by delayed eyelid opening. Furthermore, when coupled with transient hearing loss that animals normally fully recover from, very early visual experience leads to inhibitory deficits that persist into adulthood. Finally, we demonstrate a functional projection from the visual to auditory cortex that could mediate these effects.

A viral strategy for targeting and manipulating interneurons across vertebrate species.

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

Characterization of auditory synaptic inputs to gerbil perirhinal cortex.

The representation of acoustic cues involves regions downstream from the auditory cortex (ACx). One such area, the perirhinal cortex (PRh), processes sensory signals containing mnemonic information. Therefore, our goal was to assess whether PRh receives auditory inputs from the auditory thalamus (MG) and ACx in an auditory thalamocortical brain slice preparation and characterize these afferent-driven synaptic properties. When the MG or ACx was electrically stimulated, synaptic responses were recorded from the PRh neurons. Blockade of type A gamma-aminobutyric acid (GABA-A) receptors dramatically increased the amplitude of evoked excitatory potentials. Stimulation of the MG or ACx also evoked calcium transients in most PRh neurons. Separately, when fluoro ruby was injected in ACx in vivo, anterogradely labeled axons and terminals were observed in the PRh. Collectively, these data show that the PRh integrates auditory information from the MG and ACx and that auditory driven inhibition dominates the postsynaptic responses in a non-sensory cortical region downstream from the ACx.

Glycine mediated alterations in intracellular pH.

Glycinergic transmission shapes the coding properties of the lateral superior olivary nucleus (LSO). We investigated intracellular pH responses in the LSO to glycine using BCECF-AM in brain slices. With extracellular bicarbonate, glycine produced an alkalinization followed by an acidification while, in the nominal absence of bicarbonate, glycine produced acidifications. Separately, in whole-cell recordings from LSO neurons, glycine caused hyperpolarization followed by long-lasting depolarization. While the bicarbonate-dependent intracellular alkalinization could be related to chloride/bicarbonate exchange, bicarbonate-independent acidification may be triggered by depolarization.

Developmental expression of inhibitory synaptic long-term potentiation in the lateral superior olive.

Principal neurons of the lateral superior olivary nucleus (LSO) respond selectively to interaural level differences (ILD). To perform this computation, LSO neurons integrate excitatory synaptic drive from the ipsilateral ear with inhibitory synaptic drive from the contralateral ear via the medial nucleus of the trapezoid body (MNTB). Previous research demonstrated that inhibitory terminals from the MNTB to the LSO are eliminated during development. Furthermore, MNTB synapses display an activity- and age-dependent long-term depression (iLTD) that may contribute to inhibitory synapse elimination. However, inhibitory synapses that are stabilized become stronger. Here, we asked whether MNTB synapses displayed activity-dependent strengthening. Whole-cell recordings were obtained from LSO neurons in a gerbil brain slice before and after hearing onset. The inhibitory MNTB afferents were stimulated at a low rate, similar to spontaneous discharge rates observed in vivo. The MNTB-evoked inhibitory responses were strengthened by 40-300% when synaptic activity was coupled with postsynaptic membrane depolarization, exogenous glutamate application, or activation of ipsilateral excitatory synaptic inputs. This inhibitory long-term potentiation (iLTP) was associated with increased spontaneous inhibitory postsynaptic current (IPSC) amplitude and frequency. One hour after iLTP induction, IPSCs could not be de-potentiated by the MNTB stimulation pattern that induces iLTD in control slices. iLTP could only be induced after hearing onset (>P12), and was blocked in the presence of a GABAB receptor antagonist. Together, these results suggest a developmental period during which the induction of iLTP depends on the conjoint activation of GABAB receptors and postsynaptic depolarization. We propose that iLTP may support stabilization of un-pruned MNTB connections and contribute to the emergence of ILD processing in the mature LSO.

Rescue of inhibitory synapse strength following developmental hearing loss.

Inhibitory synapse dysfunction may contribute to many developmental brain disorders, including the secondary consequences of sensory deprivation. In fact, developmental hearing loss leads to a profound reduction in the strength of inhibitory postsynaptic currents (IPSCs) in the auditory cortex, and this deficit persists into adulthood. This finding is consistent with the general theory that the emergence of mature synaptic properties requires activity during development. Therefore, we tested the prediction that inhibitory strength can be restored following developmental hearing loss by boosting GABAergic transmission in vivo. Conductive or sensorineural hearing loss was induced surgically in gerbils prior to hearing onset and GABA agonists were then administered for one week. IPSCs were subsequently recorded from pyramidal neurons in a thalamocortical brain slice preparation. Administration of either a GABA(A) receptor a1 subunit specific agonist (zolpidem), or a selective GABA reuptake inhibitor (SGRI), rescued IPSC amplitude in hearing loss animals. Furthermore, this restoration persisted in adults, long after drug treatment ended. In contrast, a GABA(B) receptor agonist baclofen did not restore inhibitory strength. IPSCs could also be restored when SGRI administration began 3 weeks after sensory deprivation. Together, these results demonstrate long-lasting restoration of cortical inhibitory strength in the absence of normal experience. This suggests that in vivo GABA(A) receptor activation is sufficient to promote maturation, and this principle may extend to other developmental disorders associated with diminished inhibitory function.