Respiratory and Telencephalic Modulation of Vocal Motor Neurons in the Zebra Finch

Morphological and intrinsic electrical features of XIIts MNs. A, Intracellular staining of an XIIts MN shows dendrites extending into regions surrounding nXIIts and an axon that travels ventrally along the midline toward the XIIth nerve rootlet. Motor neurons (gray polygons) in the rostral portion of XIIts were labeled by injection of retrograde tracer into the ventral syringeal muscle several days before intracellular filling in a brain slice preparation. D, Dorsal; L, lateral. B, In vitro, XIIts MNs generated highly regular trains of action potentials in response to positive currents. Membrane potential responses of an XIIts MN to positive and negative injected currents (±200 pA; the pulse duration is shown below the superimposed voltagetraces) passed through the recording electrode are shown from an in vitro intracellular recording made in a brain slice. The inset trace highlights the spike afterdepolarization (ADP) characteristic of XIIts MNs.C, An in vivo intracellular membrane potential recorded from a XIIts MN shows that in response to a +200 pA current injected through the recording electrode, the cell generates a train of action potentials similar to those seen in vitro, but with action potential suppression, likely to be occurring during the inspiratory phase of respiration (arrow) (see Fig. 8 and Results). Note that subthreshold membrane potential movement during this inspiratory phase consists of a gradual rather than abrupt hyperpolarization. D, The average firing frequencies (in hertz; in vitro,left; in vivo, right) are plotted as a function of the amplitude of the positive current injected through the recording electrode for populations of XIIts MNs (in vitro, 40 cells; in vivo, 5 cells). The average firing frequency as a function of injected current in vitro and in vivo was highly linear between 0 and +0.6 nA. E, Instantaneous action potential frequency plots (in vitro, left; in vivo, right; averages from same cells as inD).

A schematic summarizing the types and frequencies of synaptic and antidromic responses evoked in XIIts MNs after electrical stimulation at different sites (marked byasterisks) within the transverse brain slice preparation of the caudal medulla. A, Stimulation in ipsilateral RAm predominantly evoked IPSPs and rarely evoked EPSPs. B, Contralateral RAm stimulation evoked IPSPs and EPSPs from a small fraction of the cells tested. C, D, Stimulation in the XIIts nerve root evoked antidromic spikes (ipsilateral stimulation) and IPSPs (contralateral stimulation) in XIIts MNs. The numbers in parenthesesrefer to the number of cells showing the response illustrated by the accompanying trace of the total number of cells tested.D, Dorsal; L, lateral.

Electrical stimulation in RAm evokes synaptic inhibition in XIIts MNs mediated predominantly by glycine receptors.A, IPSPs evoked in XIIts MNs by ispilateral RAm stimulation (Fig. 3A) were abolished by combined application of GABA_A and glycine receptor blockers (BMI and strychnine; see Materials and Methods for details). This treatment reversibly abolished the IPSPs elicited in XIIts MNs and unmasked EPSPs. An asterisk marks stimulation time.B, Scatterplots of PSP amplitude before (control) and immediately after treatment with strychnine and BMI (left) or strychnine alone (right).

In the presence of glycine receptor blockers, electrical stimulation in ipsilateral RAm evoked EPSPs in XIIts MNs mediated in part by NMDA receptors and which were sufficient to translate single EPSPs into a sustained action potential discharge.A, A single EPSP could trigger sustained trains of action potentials at the actual resting potential of the cell (actual Vm) but was rendered highly phasic and entirely subthreshold when the impaled XIIts MN was slightly hyperpolarized via tonic negative current injection through the recording electrode (more negative Vm). B, The slow portion of the EPSP evoked by RAm stimulation in brain slices bathed in strychnine had the voltage-dependence characteristic of NMDA receptor-mediated transmission and was strongly attenuated by hyperpolarization of the postsynaptic XIIts membrane. The EPSP evoked by RAm stimulation is displayed at the actual resting potential of the cell (topmost trace; actual Vm) or at a slightly more negative membrane potential (bottom trace;more negative Vm) achieved by passing tonic current through the recording electrode. The EPSP triggered a train of action potentials at the more positive membrane potential. Subsequent treatment with the NMDA receptor blocker d-APV reduced the EPSP in a manner resembling postsynaptic hyperpolarization, localizing the NMDA receptors to the impaled cell. C, The slow portion of the EPSP was blocked reversibly by applying NMDA receptor antagonists to XIIts. EPSPs that were collected before and during application of the NMDA receptor antagonist d-APV to XIIts are shown superimposed (see Materials and Methods). These results suggest that NMDA receptors on the XIIts MN are activated by lateral medullary inputs. Raw traces are shown in A andB, and averages of three to six traces are shown inC. Stim, Stimulation time.

Scatterplots of EPSP amplitude before (control) and with the impaled cell held in a hyperpolarized state (left; more negative V_m) or treated with d-APV (right; APV). With either treatment, EPSPs were attenuated to a similar degree (Table 2).

EPSPs and IPSPs evoked in XIIts MNs from lateral medullary stimulation are attributable to cell bodies local to RAm. Incontrol ACSF (top), electrical stimulation and glutamate application in the same spot in the lateral medulla evoked similar inhibitory responses in this XIIts MN. After strychnine application (bottom), which abolished the inhibitory responses, the same stimulation protocol evoked depolarizing, excitatory responses. An arrow marks the time of electrical (left) or chemical (glutamate; right) stimulation in each case. Traces shown are averages of three to six individual records.

XIIts motor neurons display a pronounced respiratory rhythm. An in vivo intracellular recording from an XIIts MN was used to monitor its membrane potential, whereas a simultaneous EMG was obtained from abdominal expiratory muscles to measure respiratory activity. XIIts MN firing was always in phase with the expiratory EMG.

Membrane potential manipulations reveal phasic excitation at expiratory EMG onset. An in vivointracellular recording (mV) from an XIIts MN and a simultaneous abdominal muscle EMG (EMG) were used to measure the nature of respiratory activity in XIIts. During these recordings, the XIIts MN membrane potential was either slightly depolarized (top) or moderately or more strongly hyperpolarized (middle and bottom) by injecting positive or negative currents through the recording electrode. When slightly depolarized from its resting membrane potential value with positive current (+0.5 nA, top), the neuron showed only gradual membrane hyperpolarization (but abrupt firing rate suppression) during the inspiratory phase. When moderately or more substantially hyperpolarized from its resting membrane potential with tonic negative current passed through the recording electrode (−1.25 nA, middle; −1.5 nA,bottom), the neuron displayed subthreshold excitation occurring abruptly at the onset of expiration. No membrane potential movements were noted at the offset of expiratory EMG activity.

The BOS playback-evoked responses in XIIts depend on activity in the telencephalic song nucleus HVc. A multiunit extracellular recording was made in XIIts, and baseline responses to BOS playback (shown as an oscillogram at the bottom) were collected [top,Pre-inactivation; 10 stimulus iterations; responses are shown as action potential PSTHs (XIIts spikes/bin)]. The telencephalic song nucleus HVc was then inactivated by injecting it with concentrated GABA (0.25 min 0.9% saline) ejected through a puffer pipette placed stereotactically in the nucleus. BOS playback failed to excite XIIts MNs when HVc was inactivated with GABA (middle,GABA into HVc). Reponses to BOS playback could again be detected within several minutes after GABA application to HVc (bottom, Post-inactivation). Response strengths (RS) and significance levels are given for each condition.

XIIts MNs show subthreshold and suprathreshold responses that are highly selective for forward over reverse BOS playback. A, Z-scores of forward (x-axis) and reverse BOS (REV BOS) (y-axis) evoked suprathreshold responses (from extracellular and intracellular recordings; filled circles and filled squares, respectively) and subthreshold responses (from intracellular recordings; open squares) show an overwhelming bias to the forward stimulus, indicative of strong temporal sensitivity to the BOS. All forward BOS responses were highly significant; an arrow notes the only significant reverse BOS response, which involved firing rate suppression. B, Within-cell comparisons of subthreshold versus suprathresholdd′ values obtained from intracellular records indicate that XIIts MNs are more selective for forward over reverse BOS at a suprathreshold than at the subthreshold level (dashed lines indicate the lower bounds for selective BOS responses).

Forward and not reverse playback of the bird's own song (BOS) strongly excites XIIts MNs. An in vivo intracellular recording from an XIIts MN reveals its membrane potential responses [XIIts Vm (mV)] to playback of the BOS and reverse BOS (REV BOS); a corresponding expiratory EMG is shown below each intracellulartrace. Strong excitatory suprathreshold responses accompanied by subthreshold depolarizations were detected in response to forward, but not reverse, BOS playback. Unlike XIIts activity in the absence of song playback, hyperpolarizing events can be seen (top trace, marked by an arrow at the onset of the BOS-evoked response), and strong excitation could occur out of phase with the expiratory EMG. The absence of subthreshold responses to reverse BOS (Figs. 11, 14) in XIIts indicates that its auditory afferent does not generate action potentials to this stimulus. Such behavior is characteristic of neurons in RA, the telencephalic afferent of XIIts, consistent with the idea that RA is the source of XIIts auditory responses.

Excitatory BOS-evoked responses in XIIts MNs could be preceded by subthreshold hyperpolarizations, suggestive of auditory-evoked inhibition. A, An in vivointracellular recording from an XIIts MN revealed that strong excitatory responses were evoked by BOS playback (action potential responses to the entire BOS playback are shown as a PSTH at thetop). An expanded region near BOS onset (shown as an oscillogram at the bottom) shows the PSTH and the averaged median-filtered membrane potential record aligned to the stimulus. Note that a sharp hyperpolarization precedes the initial volley of action potentials. This feature could be mediated by inhibition onto the XIIts MN driven by RA axons that innervate lateral medullary areas including RAm and PAm (Fig. 1). B, Individual, median-filtered membrane potential traces from three cells [including the cell shown in A (cell 1)] show that BOS-evoked hyperpolarizations exceeded the negativities associated with the resting respiratory rhythm (marked by a horizontal line), even when the cell was held at relatively hyperpolarized potentials with tonic negative current (cell 1 and cell 2; “resting” membrane potential in millivolts is indicated to theleft of each trace). Song onset marks the beginning of the BOS playback.

Changes in XIIts MN input impedance during BOS playback and during subsequent respiratory entrainment. Forward (bottom left) and reverse BOS (REV BOS) (bottom right) playback-evoked vocal and respiratory activity, as revealed by simultaneous abdominal expiratory EMGs (top) and intracellular recordings from an XIIts neuron [action potential PSTH (XIIts spikes) and median-filtered average membrane potential (XIIts m)]. In the lower of the two median filtered traces, the XIIts MN was injected with brief (20 msec) hyperpolarizing (−0.5 nA) currents through the recording electrode at 8 Hz to monitor postsynaptic impedance changes indicative of changes in synaptic conductances. Forward BOS strongly excited the XIIts neuron, resulting in marked impedance decreases (open arrows), consistent with elevated excitatory synaptic drive during song playback. Note that expiratory activity increased just before the augmentation in XIIts MN (enhanced EMG activity is marked by a solid arrow). Inspiration, reflected as troughs in the EMG, consistently followed forward BOS playback in this case. Such respiratory entrainment also occurred in response to reverse BOS playback, although this stimulus did not excite the XIIts neuron (note different y-axis scales for the forward vs reverse BOS-evoked XIIts PSTHs). Postsynaptic impedance decreases, measured as decrements in the amplitude of the DC-evoked hyperpolarizations, were not detected during such entrained inspiration, although slight impedance decreases were noted during subsequent expiratory phases (marked by open arrows, aligned to XIIts spiking and EMGs with closed arrows). These results are consistent with the idea that the XIIts respiratory rhythm derives primarily or solely from expiratory-driven excitation. Seventy iterations each of forward and reverse BOS playback were used to construct the PSTHs of XIIts MN and expiratory EMG activity, whereas 20 iterations were used to generate each of the two median filteredV_m values (i.e., current pulses were not injected through the recording electrode on every trial).