The HVC microcircuit: the synaptic basis for interactions between song motor and vocal plasticity pathways

Abstract

Synaptic interactions between telencephalic neurons innervating descending motor or basal ganglia pathways are essential in the learning, planning, and execution of complex movements. Synaptic interactions within the songbird telencephalic nucleus HVC are implicated in motor and auditory activity associated with learned vocalizations. HVC contains projection neurons (PNs) (HVC(RA)) that innervate song premotor areas, other PNs (HVC(X)) that innervate a basal ganglia pathway necessary for vocal plasticity, and interneurons (HVC(INT)). During singing, HVC(RA) fire in temporally sparse bursts, possibly because of HVC(INT)-HVC(RA) interactions, and a corollary discharge can be detected in the basal ganglia pathway, likely because of synaptic transmission from HVC(RA) to HVC(X) cells. During song playback, local interactions, including inhibition onto HVC(X) cells, shape highly selective responses that distinguish HVC from its auditory afferents. To better understand the synaptic substrate for the motor and auditory properties of HVC, we made intracellular recordings from pairs of HVC neurons in adult male zebra finch brain slices and used spike-triggered averages to assess synaptic connectivity. A major synaptic interaction between the PNs was a disynaptic inhibition from HVC(RA) to HVC(X), which could link song motor signals in the two outputs of HVC and account for some of the song playback-evoked inhibition in HVC(X) cells. Furthermore, single interneurons made divergent connections onto PNs of both types, and either PN type could form reciprocal connections with interneurons. In these two regards, the synaptic architecture of HVC resembles that described in some pattern-generating networks, underscoring features likely to be important to singing and song learning.

Figure 1.

The song nucleus HVC in the zebra finch telencephalon contains two different classes of PN and at least one class of interneurons. A, A sagittal section through the telencephalon of an adult male zebra finch, stained for myelin, showing the song nucleus HVC and one of its two efferent targets, the song premotor nucleus RA. HVC fibers transiting to RA can be seen between the two nuclei. Area X, the other efferent target of HVC, is medial to this plane of section. D, Dorsal; R, rostral. B, A schematic of the song nucleus HVC, showing the three neuron classes, including PNs that innervate RA (HVC_RA), PNs that innervate Area X (HVC_X), and interneurons. Simultaneous dual electrode recordings were made from different pairs of HVC neurons to study their synaptic connectivity. C, Confocal images of the three HVC neuron types studied here, as revealed by intracellular staining with Neurobiotin and post hoc visualization with avidin-Alexa Fluor 488 (see Materials and Methods). HVC_RA neurons (top) possessed slender and sparsely spinous dendrites and elaborated a main axon that exited at the caudal margin of HVC. HVC_X neurons (middle) were characterized by thicker and more spinous dendrites and an axon that exited HVC along its rostroventral border. Interneurons (bottom) were characterized by aspinous, varicose dendrites and lacked an axon that exited HVC. Scale bar, 20 μm. D, Typical membrane potential responses of the three HVC neuron types to depolarizing current pulses (bottom trace). HVC_RA neurons (top trace) fired only one or a few action potentials, even in response to large-amplitude depolarizing currents (+1.5 nA). HVC_X neurons (middle trace) fired repetitively to moderate currents (+0.5 nA) with some spike-frequency accommodation. Interneurons (HVC_INT) fired at high frequencies with little or no spike-frequency accommodation in response to moderate depolarizing current (+0.5 nA). Action potential widths of interneurons are narrower than in either of the PN types (data not shown). Resting potentials (in millivolts) are shown to the left of each membrane potential trace.

Figure 5.

Dual intracellular recordings reveal that HVC_INT provide short-latency inhibition onto HVC_X cells. A, Raw membrane potential records from a synaptically coupled interneuron (bottom) and HVC_X cell (top). DC-evoked spikes could evoke robust IPSPs in the HVC_X cell; note that a spontaneous IPSP, presumably from another interneuron, occurred after the DC-evoked responses. B, The mean STA from all HVC_INT-HVC_X cell pairs compared with the mean STA from all HVC_RA-HVC_X cells pairs, showing the offset in the 25% rise times (horizontal dashed line) (see Table 2). The overall shapes of the STAs in the different cell pairs were very similar, but the HVC_RA-HVC_X STA was delayed relative to HVC_INT-HVC_X STA, suggesting that HVC_RA cells are connected indirectly with HVC_X cells. The STA conventions are as in Figures 2 and 4. C, Higher-frequency firing in an HVC_INT can drive a sustained hyperpolarization in the HVC_X cell. D, HVC_X cells in some cases could drive EPSPs in an HVC_INT cell. DC-evoked firing in the HVC_X cell (bottom trace) reliably evoked suprathreshold EPSPs in the HVC_INT (top trace). The HVC_INT also evoked IPSPs in the HVC_X cell (shown in Fig. 9D), indicating that interneurons and HVC_X cells can form reciprocal synaptic connections.

Figure 2.

Action potentials in HVC_RA neurons evoke IPSPs in HVC_X neurons. A, Dual intracellular recordings show that DC-evoked action potentials in the HVC_RA neuron (bottom trace) can evoke IPSPs in the HVC_X neuron. B, An HVC_RA STA of the HVC_X neuron membrane potential, plotted relative to the HVC_RA action potential peak (0 ms; positive times follow the action potential), for the cell pair shown in A. A membrane hyperpolarization followed the HVC_RA action potential, indicating that HVC_RA neurons directly or indirectly drive IPSPs in the HVC_X cell. C, In some HVC_RA-HVC_X cell pairs, spike doublets in the HVC_RA neuron were necessary to drive IPSPs in the HVC_X cell (right traces), whereas single spikes failed to evoke any response (left traces), suggestive of disynaptic coupling.

Figure 3.

Antidromic stimulation of HVC_RA axons can be used to characterize the pharmacological nature of the inhibitory interactions between HVC_RA and HVC_X cells. A schematic of the slice preparation (top), showing how antidromic stimulation of the HVC_RA axon fiber bundle (lightning bolt) can be used to activate the HVC microcircuit while recording intracellularly from HVC_X cells. In this model, local collaterals of the HVC_RA axon excite HVC_INT, which ultimately drive IPSPs in the HVC_X cell. Consistent with this idea, electrical stimulation of the HVC_RA fibers drives IPSPs in HVC_X cells (bottom; control), which are blocked by the bath application of ionotropic glutamate receptor antagonists (NBQX/APV).

Figure 4.

Dual intracellular recordings provide direct evidence of the excitatory synapses that HVC_RA neurons make with HVC_INT. A, Depolarizing current pulses injected into the HVC_RA neuron (bottom trace) elicit action potentials, which were followed at short latency by subthreshold (left) and suprathreshold (right) EPSPs in the HVC_INT. B, An STA of HVC_INT membrane potential triggered off of the HVC_RA action potential, from the cell pair shown in A. A fast-rising, short-latency-positive STA was detected after the HVC_RA action potential, consistent with the idea that the HVC_RA neuron makes an excitatory synapse with the interneuron. C, Longer depolarizing currents could evoke irregular spiking in the HVC_RA neuron (bottom trace), which were paralleled by dEPSPs in the HVC_INT. In this case, note that the last six HVC_RA spikes occurred in doublets and that the second dPSP was larger than the one immediately preceding it, suggestive of synaptic facilitation.

Figure 9.

Sequential paired recordings reveal divergent and convergent inhibitory and excitatory synaptic connections in HVC. A, Sequential recordings from an HVC_RA and HVC_X neuron while maintaining an intracellular recording from an interneuron. The averages of both PN membrane potentials triggered off of the action potentials of the interneuron showed that a single interneuron could evoke IPSPs in both cells. B, Sequential recordings from three different HVC_X neurons (HVC_X 1-3) show that action potentials in a single interneuron could evoke IPSPs in all three HVC_X cells. C, Sequential recordings from an interneuron and two different HVC_RA neurons while maintaining a recording from a single HVC_X cell show that both HVC_RA cells and the interneuron can provide inhibitory input onto the same HVC_X cell. D, A reciprocally connected interneuron-HVC_X cell pair also receives synaptic input from HVC_RA axon collaterals. Antidromic stimulation of HVC_RA axons evokes an EPSP in the interneuron (top) and an IPSP in the HVC_X cell (bottom). Spike-triggered averaging reveals that the interneuron evokes an IPSP in the HVC_X cell, which in turn could evoke a dPSP in the interneuron. This is the same HVC_X-interneuron pair shown in Figure 5D, in which action potentials in the HVC_X cell evoked suprathreshold EPSPs in the interneuron. These recordings show that both HVC_X and HVC_RA axon collaterals can excite the same interneuron in HVC.

Figure 6.

The IPSPs evoked in HVC_X cells by both interneurons and HVC_RA cells were mediated by GABA_A receptors. A, In this interneuron-HVC_X cell pair, action potentials in the interneuron evoked a hyperpolarizing response in the HVC_X cell, indicative of an IPSP (control). Bath application of the GABA_A receptor antagonist PTX blocked the IPSP. B, A negative STA of the HVC_X membrane potential evoked by action potentials in an HVC_RA neuron was also blocked by the bath application of PTX. C, An IPSP evoked in an HVC_X cell by DC-evoked action potentials in a simultaneously recorded interneuron did not decrement in the presence of ionotropic glutamate receptor blockers NBQX and d-APV. D, In the same pair of neurons, antidromic stimulation of the HVC_RA fiber tract (arrow) evoked an EPSP in the interneuron (bottom; control) and an IPSP in the simultaneously recorded HVC_X cell (top; control). Subsequent bath application of NBQX/d-APV greatly reduced the excitation onto the interneuron and abolished the IPSP in the HVC_X cell. Thus, in the presence of compounds that block fast excitatory transmission, inhibition from HVC_RA onto HVC_X cells is abolished, although inhibition from the interneuron onto the HVC_X cell persists. E, Antidromic stimulation of the HVC_RA fiber tract was used to evoke an IPSP in an HVC_X cell (control). Subsequent bath application of PTX abolished the IPSP, unmasking robust EPSPs, resulting in repetitive action potential discharge in the HVC_X neuron (PTX; middle; 4 spikes in burst; mean burst rate, 111 Hz). The subsequent addition of the ionotropic glutamate receptor blockers NBQX and d-APV blocked all synaptic responses in the HVC_X cell.

Figure 7.

Blocking GABA_A-mediated inhibition in HVC could unmask additional excitatory and inhibitory synaptic pathways from HVC_RA to HVC_X neurons. A, In a PTX-treated brain slice, antidromic stimulation of HVC_RA neurons could evoke repetitive bursting (early burst: 3 spikes, 200 Hz mean burst rate; later burst: 2 spikes, 59 Hz burst rate) and slow hyperpolarizing responses, which were blocked by the bath application of the ionotropic glutamate receptor antagonists NBQX and d-APV. B, Before PTX treatment, antidromic stimulation of the HVC_RA fibers evoked a fast IPSP in an HVC_X neuron (control); subsequent PTX treatment blocked the early, fast IPSP and unmasked a multiphasic IPSP that included a slow component (PTX early). At later times during the treatment (PTX late), the same stimulation evoked an initial excitatory response, followed by a prolonged, biphasic hyperpolarization. The subthreshold depolarization that occurs during the middle of the slow, biphasic hyperpolarization is believed to be similar to the event associated with the longer-latency bursting behavior in A.

Figure 8.

Fast-spiking interneurons that evoked IPSPs in HVC_X neurons are PV+. A, Top, A single optical section of a confocal image of an intracellularly stained interneuron (green) with immunohistochemical staining for PV (red). Action potentials in this interneuron evoked IPSPs in an HVC_X cell (data not shown). In the bottom panel, only the red wavelength is shown, showing that the soma of the interneuron was PV+. B, An HVC_X neuron (green) that received inhibitory input from a fast-spiking interneuron; a PV+ cell body was closely apposed to the HVC_X neuron soma.

Figure 10.

The major synaptic features of the HVC microcircuit revealed in the present study by paired recordings and antidromic stimulation of HVC_RA neurons are shown. HVC_RA (gray circles) and HVC_X (white circles) neurons form excitatory synaptic connections (arrows) on interneurons (black circles), which provide divergent inhibitory input (t-endings) on PNs of both types. Fast excitation is mediated by ionotropic glutamate receptors, whereas fast inhibition is mediated by GABA_A receptors. Additional polysynaptic and possibly monosynaptic excitatory pathways and polysynaptic inhibitory pathways also provide a synaptic linkage from HVC_RA to HVC_X neurons (dashed lines). These monosynaptic and polysynaptic pathways are dependent on ionotropic glutamate receptors, presumably involving direct synapses between HVC_RA axon collaterals and HVC_X neurons and intervening synapses between HVC_RA axon collaterals and other HVC interneurons.