Lesions of an avian forebrain nucleus that disrupt song development alter synaptic connectivity and transmission in the vocal premotor pathway

Abstract

The avian forebrain nucleus, the lateral magnocellular nucleus of the anterior neostriatum (LMAN), is necessary for normal song development because LMAN lesions made in juvenile birds disrupt song production but do not disrupt song when made in adults. Although these age-limited behavioral effects implicate LMAN in song learning, a potential confound is that LMAN lesions could disrupt normal vocal motor function independent of any learning role by altering LMAN's premotor target, the song nucleus, the robust nucleus of the archistriatum (RA). To date, however, no studies have examined directly the effects of LMAN lesions on the circuitry of the RA. We report here that juvenile LMAN lesions rapidly and profoundly affect RA, altering synaptic connectivity within this nucleus, including descending inputs from the song nucleus HVc. Specifically, morphological assays of the dendritic spines of RA projection neurons and axon terminal boutons arising from HVc show a numerical decline in the density of connections in RA in LMAN-lesioned juveniles compared with controls. Concurrently, LMAN lesions alter excitatory transmission within the juvenile RA: after LMAN lesions, the stimulus-response relationship between HVc fibers and RA neurons steepens, and the amplitude of spontaneous monophasic EPSCs increases. Rather than arresting RA in a juvenile state, LMAN lesions transform the structure and function of RA and its connections, such that it is distinct from that of the normal juvenile. In many ways, RA circuitry in LMAN-lesioned juveniles resembles that of normal adults, suggesting that LMAN lesions induce a premature maturation of the vocal motor pathway, which may lead to a loss of behavioral plasticity and abnormal song development.

Fig. 1.

A, A simplified schematic diagram of the brain nuclei that constitute the avian song system (sagittal view). The vocal motor pathway (black) includes (in descending order) the nucleus HVc (used here as a proper name), the robust nucleus of the archistriatum (RA), the tracheosyringeal portion of the hypoglossal nucleus (nXIIts), the nucleus ambiguus (nAM), the nucleus retroambigualis (nRAm) (Nottebohm et al., 1976, 1982; Vicario, 1993;Wild, 1993a,b), and the musculature of the syrinx (the main vocal organ), larynx, and respiratory apparatus (Nottebohm et al., 1976; Wild, 1993a,b). Lesions of any of these nuclei disrupt the production of learned song (Nottebohm et al., 1976; Simpson and Vicario, 1990), and chronic recording experiments have shown premotor activity preceding the onset of song in HVc and RA (McCasland, 1987; Yu and Margoliash, 1996). The anterior forebrain pathway (open symbols, dashed arrows) is critical for normal song development and consists of area X, the medial nucleus of the dorsolateral thalamus (DLM), and the lateral magnocellular nucleus of the anterior neostriatum (LMAN) (Okuhata and Saito, 1987; Bottjer et al., 1989). The primary input to the anterior forebrain pathway is from HVc to area X; the sole output of LMAN to the vocal motor pathway is onto RA. B, Camera lucida drawings of representative RA projection neurons at the three developmental stages studied here (fledgling, juvenile, and adult, on the right), as well in LMAN-lesioned juveniles (left), show the increased dendritic spine density of the normal juvenile cells relative to other ages and treatments. The spine densities of the two juvenile cells shown here are marked byarrows in Figure 3B.

Fig. 2.

The morphology of RA projection neurons changed markedly over development, and juvenile cells were larger and more complex than their fledgling and adult counterparts. A, Dendritic spine densities (black) were significantly higher in juvenile RA projection cells than in fledglings (p<.001¹) or adults (p<.03¹), and whereas adult spine density remained higher than in fledglings (p<.04¹). The soma areas (white) of juvenile RA neurons were larger than those of fledglings (p<.002²).B, The total dendritic length of RA projection neurons (black) was quite stable over development, in contrast to the total length of their local axon collaterals, which doubled between fledgling and juvenile times (p<.0001³) and then declined by approximately one-third by adulthood (p<.04²).C, Sholl analysis indicated that the more distal dendritic arbor (i.e., 80–120 μm from the cell body) increased in complexity (i.e., number of intersections) between fledgling and juvenile times; these differences are maintained in the adult. Two-way ANOVAs confirmed this result: there was a significant overall effect of age on number of dendritic intersections between fledglings and juveniles [p < 0.05; there was also a significant (p < 0.05) interaction between age and radius] and between fledglings and adults (p < 0.03), but not between juveniles and adults. D, A similar analysis of axon collateral complexity showed that juvenile RA cells had more complex local collaterals than their fledgling or adult counterparts. The increase in complexity from fledglings to juveniles occurred across the entire extent of the axon collateral arbor (40–140 μm), but the decrease between juveniles and adults was found only in the distal-most portion of the arbor (120–140 μm). Two-way ANOVAs showed that age had a significant overall effect on collateral intersections between juveniles and both fledglings (p < 0.0001) and adults (p < 0.001). There were no significant differences in local axonal complexity between adults and fledglings at any individual radius, but a two-way ANOVA did show an overall effect of age (p < 0.03), suggesting that adult RA projection neurons retained somewhat higher local collateral complexity than in fledglings. Statistically significant p values: *juvenile (Juv) versus fledgling (Fledge); **juvenile versus adult (Adult); ***adult versus fledgling. Statistical tests:¹Mann–Whitney U test (1 or more of the distributions was significantly non-normal by the Shapiro-Wilk test);²two-tailed Student's t test assuming equal variance (both distributions normal); ³two-tailed Student's t test assuming unequal variance (both distributions normal; variances significantly unequal by one or more of the O'Brien, Brown-Forsythe, Levene, or Bartlett tests). Fledgling:n = 15 cells from eight birds for spine density and soma distributions, 14 cells from eight birds for all dendrite and axon collateral comparisons. Juvenile: n = 15 cells from nine birds; adult: n = 13 cells from nine birds.

Fig. 3.

Juvenile LMAN lesions induced rapid morphological changes in RA projection neurons. A, The dendritic spine density (gray) and soma area (white) of RA neurons in LMAN-lesioned juvenile birds was lower than in age-matched controls (lesions were made 4–7 d before data collection). B, The dendritic spine densities of RA neurons from juvenile birds with LMAN lesions (black bars) fell into two distinct classes, one much lower than those of control cells (open bars), and another population that was indistinguishable from control values. The individual cells are presented in random order along the x-axis, except that cells from lesioned and control birds, as well as the higher and lower spine density classes of cells from lesioned birds, are grouped separately. The spine density distribution in lesioned birds was significantly non-normal (p < 0.04, Shapiro-Wilk test). Arrows mark values obtained from the two juvenile RA projection cells depicted in Figure 1B.C, The dendritic complexity of juvenile RA projection neurons was not significantly altered by LMAN lesions, except at the most distal point measured (140 μm from the cell body). A two-way ANOVA showed no overall effect of LMAN lesions on dendritic intersections. D, The local axon collateral complexity was lower in lesioned animals, with a significant reduction at the 100 μm radius. In addition, a two-way ANOVA showed a significant (p < 0.05) interaction between lesioning and radius on collateral intersections; i.e., LMAN lesions influenced where radially the axon collaterals tend to occur, with fewer branches in the distal portion of the arbor in lesioned birds. Statistical tests: ¹Mann–Whitney U test;²two-tailed Student's t test assuming equal variance; ³two-tailed Student's t test assuming unequal variance. LMAN-lesioned juveniles:n = 16 cells from eight birds; control juveniles:n = 15 cells from nine birds.

Fig. 4.

Juvenile, but not adult, LMAN lesions reduced HVc axon terminal bouton frequency in RA. Linear bouton frequency in RA was measured (in number of boutons per micrometer of axon collateral length) after unilateral lesion of LMAN and bilateral tracer injection into HVc (see Materials and Methods). In juveniles (▪), between-hemisphere, within-bird comparisons revealed consistently lower bouton frequency in RA on the side receiving the LMAN lesion (p < 0.02, paired t test,n = 7 birds). In adults (■), on the other hand, lesions did not affect HVc bouton frequency in RA (p > 0.8, paired t test,n = 4 birds), which was higher on the lesioned side just as often as it was lower.

Fig. 5.

The stimulus–response relationship of EPSPs evoked in RA by electrical stimulation of HVc fibers in brain slices changed both with development and after LMAN lesions. A, The onset slope and amplitude of EPSPs evoked in an adult RA projection neuron (top traces) by HVc fiber stimulation (at point marked by arrow) increased as a function of current amplitude (stimulus levels are shown above each trace). Currents above 40 μA evoked an EPSP from this RA projection neuron; higher currents elicited increasingly larger EPSPs, with steeper onset slopes. At even higher stimulus intensities, these EPSPs triggered action potentials, distinguishing them as excitatory (data not shown). Measurements of the initial slope of the EPSP at different stimulus currents were used to plot the stimulus–response relationship for this cell (bottom graph), for which the two best linear fits of the stimulus–response data are superimposed (see Materials and Methods).B, The average stimulus–response relationship (i.e., the mean of the two best linear fits of the stimulus–response data) is shown for HVc EPSPs recorded from RA projection neurons in juvenile control animals (n = 12 cells from 4 birds), juveniles with LMAN lesions (n = 13 cells from 5 birds), and adults (n = 8 cells from 3 birds). The stimulus–response relationship was significantly steeper both in juveniles receiving LMAN lesions (p < 0.01) and in adults (p < 0.05), relative to control juveniles (Mann–Whitney U test).C, D, Stimulus–response plots show all of the individual EPSP slope measurements made from each cell in juvenile control slices (C, D), juveniles with LMAN lesions (C), and adults (D). Responses are plotted against stimulation current normalized to threshold, i.e., the absolute current minus the minimum current needed to evoke an EPSP for a given cell. Aline depicting the average stimulus–response relationship for each cell class is superimposed on these individual points. Note that the juvenile control cells yield many slope measurements with a shallower relationship to the stimulus current than was seen in cells from juveniles with LMAN lesions or adults.

Fig. 6.

Unilateral LMAN lesions altered the amplitude distribution and types of spontaneous synaptic currents recorded in RA in brain slices made from juvenile animals. A,Top, Current records obtained in whole-cell voltage-clamp mode (V_h = −60 mV) from RA neurons from either control (left) or LMAN-lesioned (right) hemispheres, in the presence of 50 μm picrotoxin. Bottom, Representative large and small synaptic currents are shown on an expanded time scale (the upper and lower values next to the calibration bars correspond to the upper and lower current records, respectively). In control conditions, large amplitude, polyphasic currents occurred periodically, along with smaller amplitude, monophasic currents. In recordings from RA neurons in slices from the LMAN-lesioned hemisphere, only monophasic currents were observed; these varied in amplitude, and >97.5% of them were <30 pA. B, A frequency histogram of all of the spontaneous synaptic currents recorded from RA neurons from control hemispheres, versus currents obtained from RA cells in LMAN-lesioned hemispheres. Note that the peak of the distribution for the events in “lesioned” cells is to the right of that for the control events, and that very large amplitude currents (i.e., >100 pA) were detected only in control hemispheres. These larger events were the polyphasic currents shown on the left inA, whereas smaller events (i.e., <30 pA; to theleft of the arrow in B) were consistently monophasic. C, Cumulative amplitude distributions of monophasic EPSCs (<30 pA) show a rightward shift in events measured in lesioned cells relative to controls. These distributions were significantly different (p ≪ 0.0001, Kolmogorov–Smirnov test).