Estradiol impacts a wide variety of brain processes, including sex differentiation, mood, and learning. Here we show that estradiol regulates auditory processing of acoustic signals in the vertebrate brain, more specifically in the caudomedial nidopallium (NCM), the songbird analog of the mammalian auditory association cortex. Multielectrode recordings coupled with local pharmacological manipulations in awake animals reveal that both exogenous and locally generated estradiol increase auditory-evoked activity in NCM. This enhancement in neuronal responses is mediated by suppression of local inhibitory transmission. Surprisingly, we also found that estradiol is both necessary and sufficient for the induction of multiple mitogen-activated protein kinase (MAPK)-dependent genes thought to be required for synaptic plasticity and memorization of birdsong. Specifically, we show that local blockade of estrogen receptors or aromatase activity in awake birds decrease song-induced MAPK-dependent gene expression. Infusions of estradiol in acoustically isolated birds induce transcriptional activation of these genes to levels comparable with song-stimulated animals. Our results reveal acute and rapid nongenomic functions for estradiol in central auditory physiology and suggest that such roles may be ubiquitously expressed across sensory systems.

Glutamate uptake by high-affinity transporters is responsible for limiting the activation of postsynaptic receptors and maintaining low levels of ambient glutamate. The reuptake process generates membrane currents, which can be activated by synaptically released glutamate in glial cells and some postsynaptic neurons. However, less is known about presynaptic transporter currents because the small size of synaptic boutons precludes direct recordings. Here, we have recorded from two giant nerve terminals: bipolar cell synaptic terminals in goldfish retina and the calyx of Held in rat auditory brainstem. Exocytosis was evoked by brief depolarizations and measured as an increase in membrane capacitance. In isolated bipolar cell terminals, exocytosis was associated with an anion (NO3- or Cl-) current. The current peaked 2.8 msec after the start of the depolarization and decayed with a mean time constant of 8.5 msec. It was inhibited by the nontransportable glutamate transporter antagonist sc-threo-beta-benzyloxyaspartate (TBOA) but was insensitive to the GLT1/EAAT2 subtype-selective antagonist dihydrokainate and was affected by extracellular pH buffering. A TBOA-sensitive anion current was also evoked by application of exogenous glutamate to bipolar cell terminals. The large single-channel conductance, derived from noise analysis, and previous immunolocalization studies suggest that synaptically released glutamate activates EAAT5-type transporters in bipolar cell terminals. In contrast, neither exocytosis nor exogenous glutamate evoked a transporter current in the calyx of Held. Glutamate transporter currents with rapid kinetics are therefore identified and characterized in bipolar cell terminals, providing a valuable system for investigating the function and modulation of presynaptic glutamate transporters.

Auditory processing and hearing-related pathologies are heavily influenced by steroid hormones in a variety of vertebrate species, including humans. The hormone estradiol has been recently shown to directly modulate the gain of central auditory neurons, in real time, by controlling the strength of inhibitory transmission via a nongenomic mechanism. The functional relevance of this modulation, however, remains unknown. Here we show that estradiol generated in the songbird homolog of the mammalian auditory association cortex, rapidly enhances the effectiveness of the neural coding of complex, learned acoustic signals in awake zebra finches. Specifically, estradiol increases mutual information rates, coding efficiency, and the neural discrimination of songs. These effects are mediated by estradiol's modulation of both rate and temporal coding of auditory signals. Interference with the local action or production of estradiol in the auditory forebrain of freely behaving animals disrupts behavioral responses to songs, but not to other behaviorally relevant communication signals. Our findings directly show that estradiol is a key regulator of auditory function in the adult vertebrate brain.

The role of GABA in the central processing of complex auditory signals is not fully understood. We have studied the involvement of GABA A-mediated inhibition in the processing of birdsong, a learned vocal communication signal requiring intact hearing for its development and maintenance. We focused on caudomedial nidopallium (NCM), an area analogous to parts of the mammalian auditory cortex with selective responses to birdsong. We present evidence that GABA A-mediated inhibition plays a pronounced role in NCM's auditory processing of birdsong. Using immunocytochemistry, we show that approximately half of NCM's neurons are GABAergic. Whole cell patch-clamp recordings in a slice preparation demonstrate that, at rest, spontaneously active GABAergic synapses inhibit excitatory inputs onto NCM neurons via GABA A receptors. Multi-electrode electrophysiological recordings in awake birds show that local blockade of GABA A-mediated inhibition in NCM markedly affects the temporal pattern of song-evoked responses in NCM without modifications in frequency tuning. Surprisingly, this blockade increases the phasic and largely suppresses the tonic response component, reflecting dynamic relationships of inhibitory networks that could include disinhibition. Thus processing of learned natural communication sounds in songbirds, and possibly other vocal learners, may depend on complex interactions of inhibitory networks.

Birdsong is a learned vocal behaviour that requires intact hearing for its development in juveniles and for its maintenance during adulthood. However, the functional organization of the brain circuits involved in the perceptual processing of song has remained obscure. Here we provide evidence that GABAergic mechanisms are an important component of these circuits and participate in the auditory processing of birdsong. We first cloned a zebra finch homologue of the gene encoding the 65-kDa isoform of glutamic acid decarboxylase (zGAD-65), a specific GABAergic marker, and conducted an expression analysis by in situ hybridization to identify GABAergic cells and to map their distribution throughout auditory telencephalic areas. The results showed that field L2, the caudomedial nidopallium (NCM) and the caudomedial mesopallium (CMM) contain a high number of GABAergic cells. Using patch-clamp brain slice recordings, we found abundant GABAergic mIPSCs in NCM. Pharmacological antagonism of mIPSCs induced large EPSC bursts, suggesting that tonic inhibition helps to stabilize NCM against runaway excitation via activation of GABA-A receptors. Next, using double fluorescence in situ hybridization and double immunocytochemical labelling, we demonstrated that large numbers of GABAergic cells in NCM and CMM show inducible expression of the transcriptional regulator ZENK in response to song auditory stimulation. These data provide direct evidence that GABAergic neurons in auditory brain regions are activated by song stimulation. Altogether, our results suggest that GABAergic mechanisms participate in auditory processing and perception, and might contribute to the memorization of birdsong.

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d.

The effect of antagonism of GABA(A) receptors on the receptive fields of raccoon primary somatosensory cortical neurons was tested using microiontophoretic administration of bicuculline methiodide (BMI). The size of cutaneous receptive fields was examined using minimal suprathreshold mechanical stimulation before, during, and after BMI administration. In 65 of 102 rapidly adapting neurons, BMI produced a clear expansion of the receptive field. The mean increase in receptive-field size was 286%. The receptive fields on the distal digit, which were initially smaller, showed smaller increases in absolute area than more proximal receptive fields, but the percentage increase did not vary with location. Greater expansion was seen in superficially located neurons than in those below 800 microm. Of particular significance was the finding that the expansion of receptive fields produced by BMI never extended from one digit onto an adjacent digit or onto the palm, even when the original receptive field was at the base of a digit. This finding indicates that intracortical GABAergic inhibition is insufficient to explain cortical reorganization following digit amputation.

Cortical reorganization was induced by amputation of the 4th digit in 11 adult raccoons. Animals were studied at various intervals, ranging from 2 to 37 wk, after amputation. Recordings were made from a total of 129 neurons in the deafferented cortical region using multibarrel micropipettes. Several types of receptive fields were described in reorganized cortex: restricted fields were similar in size to the normal receptive fields in nonamputated animals; multi-regional fields included sensitive regions on both adjacent digits and/or the underlying palm and were either continuous over the entire field or consisted of split fields. The proportion of neurons with restricted fields increased with time after amputation and was greater than previously found in subcortical regions. A GABA(A) receptor antagonist (bicuculline methiodide), glutamate, and GABA were administered iontophoretically to these neurons while determining their receptive fields and thresholds. Bicuculline administration resulted in expansion of the receptive field in 60% of the 93 neurons with cutaneous fields. In most cases (33 neurons) this consisted of a simple expansion around the borders of the predrug receptive field, and the average expansion (426%) was not different from that seen in nonamputated animals. In some neurons (n = 4), bicuculline produced an expansion from one digit onto the adjacent palm or another digit, an effect never seen in control animals. Bicuculline also changed the split fields of seven neurons into continuous fields by exposing a responsive region between the split fields. Finally, bicuculline changed the internal receptive field organization of 10 neurons by revealing subfields with reduced thresholds. In contrast to the situation in nonamputated animals, iontophoretic administration of glutamate also produced receptive field expansion in some neurons (n = 6), but the size and/or shape of the change was different from that produced by bicuculline, indicating that the effects of bicuculline were not due to an overall facilitation of neuronal activity. These results are consistent with the hypotheses that an important component of long-term cortical reorganization is the gradual reduction in effective receptive field size and that intracortical inhibitory networks are partially responsible for these changes.