Neuron-specific cholinergic modulation of a forebrain song control nucleus

Abstract

Cholinergic activation profoundly affects vertebrate forebrain networks, but pathway, cell type, and modality specificity remain poorly understood. Here we investigated cell-specific cholinergic modulation of neurons in the zebra finch forebrain song control nucleus HVC using in vitro whole cell recordings. The HVC contains projection neurons that exclusively project to either another song motor nucleus RA (robust nucleus of the arcopallium) (HVC-RAn) or the basal ganglia Area X (HVC-Xn) and these populations are synaptically coupled by a network of GABAergic interneurons. Among HVC-RAn, we observed two physiologically distinct classes that fire either phasically or tonically to injected current. Muscarine excited phasic HVC-RAn and most HVC-Xn. Effects were observed under conditions of blockade of fast synaptic transmission and were reversed by atropine. In contrast, unlike what is commonly observed in mammalian systems, HVC interneurons were inhibited by muscarine and these effects were reversed by atropine. Thus cholinergic modulation reconfigures the HVC network in a more complex fashion than that implied by monolithic "gating." The two projection pathways are decoupled through suppression of the inhibitory network that links them, whereas each is simultaneously predominantly excited. We speculate that fluctuating cholinergic tone in HVC could modulate the interaction of song motor commands with basal ganglia circuitry associated with song perception and modification. Furthermore, if the in vitro distinction between RA-projecting neurons that we observed is also present in vivo, then the song system motor pathway exhibits greater physiological diversity than has been commonly assumed.

Fig. 1.

Physiological classes of HVC neurons recorded in vitro. A: responses of 4 HVC cells to near-threshold depolarizing current injection (tonic HVC-RAn [motor nucleus RA (robust nucleus of the arcopallium)]: +0.4 nA; all others: +0.2 nA), showing features characteristic of the 4 cell types we observed. There are notable differences in spike morphology, firing rates, and accommodation. Each cell here identified as a projection neuron was filled with Neurobiotin and visibly elaborated an axon that exited HVC in the direction of the efferent target nucleus. The interneuron was also filled; it had no extrinsically projecting axon and was morphologically consistent with known HVC-In (interneuron type). B: mean current-firing plots for each cell type shown in A.

Fig. 2.

Distinguishing physiological characteristics of anatomically identified HVC cell types. A: identification of phasic HVC-RAn. A histogram for all cells of the phasicness of the suprathreshold response to somatic current injection (see methods) reveals 2 modes: one highly phasic and one tonic. All of the filled cells with axons in the phasic mode (>0.5) projected to RA. Note that a distinct population of filled RA-projecting neurons clustered near zero in the tonic mode. B: identification of putative HVC-In. A histogram of normalized firing rate for all cells shows that all of the filled interneurons had values >225 normalized spikes·s⁻¹·nA⁻¹, but all projection neurons had values <225 normalized spikes·s⁻¹·nA⁻¹. C: a scatterplot of HVC-Xn (basal ganglia Area X) and tonic HVC-RAn on 2 measures of afterhyperpolarization (AHP) timing: time to peak (AHPttp) and time to decay (AHPttd) (see methods), showing relative separation between the 2 cell classes. Filled points indicate cells that were verified to have axons projecting to Area X (blue) or to RA (red). The green line was used to conservatively identify a region containing most HVC-Xn, while excluding HVC-RAn (with 2 notable exceptions).

Fig. 3.

Phasic HVC-RAn are directly excited by muscarine. A: typical response of a phasic HVC-RAn to muscarine and atropine. Panels depict ongoing measurements of evoked spike rate (top left) and membrane potential (V_m, bottom left) taken regularly throughout the recording (see methods). Horizontal bars denote the times of drug application. As indicated, this experiment was performed in the presence of fast synaptic blockers. The right panels (1–3) show sample voltage sweeps in response to +0.13-nA current pulses before (1) and after (2) wash-in of 10 μM muscarine and after subsequent addition of 1 μM atropine (3). Numbers show how the traces correspond to the time points in the plots to the left. B: distribution of changes in spiking for phasic HVC-RAn in response to cholinergic agents. White and black circles denote recordings made, respectively, in the absence and presence of synaptic blockers. For muscarine experiments, changes in spike rate for each point are calculated as the difference between the predrug and postdrug values for that experiment (see methods). For atropine, the changes are also calculated as the difference between the predrug and postdrug values, relative to values measured in the presence of muscarine. Horizontal bars denote the population means. C: distribution of changes in V_m for phasic HVC-RAn in response to cholinergic agents. This panel is organized and the data were calculated as in B.

Fig. 4.

HVC-Xn are directly excited by muscarine. A: typical response of an anatomically identified HVC-Xn to muscarine. Panels depict ongoing measurements of evoked spike rate (top left) and V_m (bottom left) measurements taken every 10 s throughout the recording (see methods). Horizontal bars denote the times of drug application. As indicated, this experiment was performed in the presence of fast synaptic blockers. The right panels (1–3) show sample voltage sweeps in response to +0.25-nA current pulses before (1) and after (2) wash-in of 10 μM muscarine and after subsequent washout of all drugs (3). Numbers show how the traces correspond to the time points in the plots to the left. B: typical response of an HVC-Xn to muscarine and atropine. Panels are organized as in A. The right panels (1–3) show sample voltage sweeps in response to +0.16-nA current pulses before (1) and after (2) wash-in of 10 μM muscarine in the presence of fast synaptic blockers and after subsequent addition of 1 μM atropine (3). C: distribution of changes in spiking for HVC-Xn in response to cholinergic agents. White and black circles denote recordings made, respectively, in the absence and presence of synaptic blockers. For muscarine and carbachol experiments, changes in spike rate for each point are calculated as the difference between the predrug and postdrug value for that experiment (see methods). For atropine, the changes are also calculated as the difference between the predrug and postdrug values, that is relative to values measured in the presence of muscarine. Horizontal bars denote the population means. D: distribution of changes in V_m for HVC-Xn in response to cholinergic agents. This panel is organized as in C.

Fig. 5.

HVC interneurons are inhibited by muscarine. A: typical response of an HVC-In to muscarine and atropine. Panels depict ongoing measurements of evoked spike rate (top left) and V_m (bottom left) measurements taken every 10 s throughout the recording (see methods). Horizontal bars denote the times of drug application. The right panels (1–3) show sample voltage sweeps in response to +0.15-nA current pulses before (1) and after (2) wash-in of 10 μM muscarine and after subsequent addition of 1 μM atropine (3). Numbers show how the traces correspond to the time points in the plots to the left. B: distribution of changes in spiking for HVC-In in response to cholinergic agents. For muscarine experiments, changes in spike rate for each point are calculated as the difference between the predrug and postdrug values for that experiment (see methods). For atropine, the changes are also calculated as the difference between the predrug and postdrug values, relative to values measured in the presence of muscarine. Changes indicated for atropine are relative to values measured after muscarine application. Horizontal bars denotes the population means. C: distribution of changes in V_m for HVC-In in response to cholinergic agents. This panel is organized as in B.