Selective innervation of neostriatal interneurons by a subclass of neuron in the globus pallidus of the rat

Abstract

A subpopulation of neurons in the globus pallidus projects to the neostriatum, which is the major recipient of afferent information to the basal ganglia. Given the moderate nature of this projection, we hypothesized that the pallidostriatal projection might exert indirect but powerful control over principal neuron activity by targeting interneurons, which comprise only a small percentage of neostriatal neurons. This was tested by the juxtacellular labeling and recording of pallidal neurons in combination with immunolabeling of postsynaptic neurons. In addition to innervating the subthalamic nucleus and output nuclei, 6 of 23 labeled pallidal neurons projected to the neostriatum. Both the firing characteristics and the extent of the axonal arborization in the neostriatum were variable. However, light and electron microscopic analysis of five pallidostriatal neurons revealed that each neuron selectively innervated neostriatal interneurons. A large proportion of the boutons of an individual axon (19-66%) made contact with parvalbumin-immunoreactive interneurons. An individual parvalbumin-immunoreactive neuron (n = 27) was apposed on average by 6.7 boutons (SD = 6.1) from a single pallidal axon (n = 2). Individual pallidostriatal boutons typically possessed more than one symmetrical synaptic specialization. In addition, 3-32% of boutons of axons from four of five pallidal neurons contacted nitric oxide synthase-immunoreactive neurons. Descending collaterals of pallidostriatal neurons were also found to make synaptic contact with dopaminergic and GABAergic neurons of the substantia nigra. These data imply that during periods of cortical activation, individual pallidal neurons may influence the activity of GABAergic interneurons of the neostriatum (which are involved in feed-forward inhibition and synchronization of principle neuron activity) while simultaneously patterning neuronal activity in basal ganglia downstream of the neostriatum.

Fig. 1.

Reconstruction along the rostrocaudal axis of the axon (black; 1 connects to2) and soma and dendrites (gray) of a single GP neuron [neuron 9678 (Table 1)] that projects to the neostriatum (NS) in addition to other basal ganglia nuclei. Note the extensive axonal arborization in the neostriatum (1353 boutons) that arises from four branches of the main axon. Boutons were located in 27 sagittal sections of 50 μm thickness. Note also the local collaterals in the GP (315 boutons) that extend well beyond the dendrites of the parent neuron. This cell also innervated the entopeduncular nucleus (EP) (27 boutons), the subthalamic nucleus (STN) (66 boutons), and the substantia nigra (247 boutons). In the substantia nigra the axon gave rise to three arborizations at three rostrocaudal levels. The most rostral arborization was restricted to the rostral part of substantia nigra pars compacta (SNc) (29 boutons). The two more caudal arborizations were located in the substantia nigra pars reticulata (SNr) (87 and 131 boutons). Scale bar, 300 μm.

Fig. 2.

Reconstruction along the rostrocaudal axis of the axon (black) and the soma and dendrites (gray) of a representative example of the majority of GP neurons, i.e., those that do not project to the neostriatum. These neurons give rise to local axonal arborizations (145 boutons in this example) and project to the entopeduncular nucleus (EP) (43 boutons in this example), the subthalamic nucleus (STN) (181 boutons in this example), and the substantia nigra (SN) (120 boutons in this example). Scale bar, 300 μm.

Fig. 3.

Physiological recordings from the GP of anesthetized rats. A, Triggered spike train of neuron 9665 (Table 1). This neuron possessed a relatively low mean firing rate and discharged in a bursting pattern. B, Triggered spike train of neuron 9672 (Table 1). This neuron discharged at a relatively high mean rate in an irregular, nonbursting pattern. C, Representative example of the modulation of spontaneous firing of a neuron in the GP during a successful juxtacellular injection. Note the alternate 200 msec periods of higher-frequency firing during current injection and lower-frequency firing when current injection was ceased. The onset and termination of current injection can be noted from the large switching artifact, which is of greater amplitude than the extracellularly recorded waveform.

Fig. 4.

A, B, Reconstructions along the rostrocaudal axis of the axon (black) and the soma and dendrites (gray) of two GP neurons (A, neuron 9666; B, neuron 9672) that project to the neostriatum in addition to other basal ganglia nuclei. Neuron 9666 gave rise to 942 boutons, and neuron 9672 gave rise to 329 boutons in the neostriatum. Note the heterogeneous nature of the local and neostriatal axonal arborizations in terms of their dimensions and density (also see Fig. 1). The arrow indicates the branch of the axon that gave rise to descending projections to the EP, STN, and SNr. Scale bar (shown in A for Aand B): 300 μm.

Fig. 5.

Light micrographs illustrating juxtacellularly labeled GP neurons (A, F) and their axons in the neostriatum (NS) in relation to PV-IR (B, D, E) and NOS-IR (C, G) interneurons. InA–E, the GP neurons were visualized with Ni-DAB giving a blue-black stain, the PV-IR interneurons were visualized with DAB and stained brown, and the NOS-IR interneurons were visualized with VIP and stainedpurple. In F and G, another combination of chromogens was used: GP neurons were visualized with DAB (brown), PV-IR neurons were visualized with Ni-DAB (blue-black), and NOS-IR interneurons were visualized with Vector VIP (purple).A, F, Juxtacellularly labeled neurons in the GP in sections that were also stained to reveal PV-IR neurons. Note the Golgi-like labeling of the filled single neurons and the many PV-IR structures. In F the neuron is located on the rostral-dorsal border of the GP, which is clearly defined by high density of PV-IR neurons in the GP compared with the lower density of PV-IR neurons in the neostriatum (NS). B, D, E, Selective innervation of PV-IR interneurons of the neostriatum by GP neurons. Note the clusters of axonal varicosities (arrows) apposing restricted parts of the PV-IR postsynaptic cells. C, D, G, Examples of the innervation of NOS-IR interneurons of the NS by the GP. C, E, The typical arrangement of multiple pallidal axonal boutons apposing individual postsynaptic neurons (arrows).D, A single GP axon forms multiple appositions with a PV-IR neuron (arrows), and in addition apposes a small-diameter dendrite of a NOS-IR interneuron (most ventral arrow). Scale bars (shown in A forA and F): 20 μm;B, 20 μm; (shown in C for C–E, G): 10 μm.

Fig. 6.

A–H, Light and electron micrographs illustrating the selective innervation of neostriatal interneurons by individual neurons of the GP. In these micrographs the axon of the GP neuron 9678 is revealed with Ni-DAB, the PV-IR structures are revealed with DAB, and NOS-immunoreactive structures are revealed with BDHC. A, Light microscopic montage of a PV-IR neuron and a NOS-IR dendrite. The PV-IR neuron is apposed by five boutons of the pallidostriatal neuron, two (b1, b2) of which contact the proximal dendrite, and three (b3–b5) of which contact the perikaryon. The NOS-IR dendrite is also apposed by a bouton (b6). B, Electron micrograph of part of the same region shown in A. The soma of the PV-IR neuron, the capillary (c), and the pallidostriatal bouton (b2) act as registration marks between the two levels of investigation. C–H, High-magnification electron micrographs of the pallidostriatal boutons in contact with the PV-IR (C–G) and NOS-IR (H) interneurons. Each bouton forms symmetrical synaptic contact (arrows) with its postsynaptic target. Some boutons possess multiple release sites (b2, b3, b5). At higher magnification the amorphous nature of the NiDAB and DAB reaction products (C–H) and the crystalline nature of the BDHC reaction product (H) are apparent. Scale bars (shown in A for Aand B): 10 μm; (shown in C forC, D–H): 0.5 μm.

Fig. 7.

Light and electron micrographs illustrating the selective innervation of NOS-IR interneurons by an individual GP neuron 9678. A, Light microscopic montage of the dendrites of two NOS-IR interneurons (visualized with BDHC). One is apposed by one bouton (b1), and the other is apposed by five boutons (b2–b6) of a densely labeled pallidostriatal axon visualized with Ni-DAB. B, Low-magnification electron micrograph illustrating part of the region inA. Note that b3 and b4 act as registration marks and b3 apposes a branch of the dendrite to which b4 is apposed. C–H, High-magnification electron micrographs of the boutons (b1–b6), illustrating their synaptic contacts with the two NOS-IR dendrites. Each bouton forms symmetrical synaptic contact with the NOS-IR dendrites identified by the BDHC reaction product (visible in C, D, andG). Note that individual boutons may possess multiple release sites (E–G). The amorphous nature of the Ni-DAB product (B–H) and the crystalline nature of the BDHC reaction product are apparent (C, F, G). Scale bars: A, 10 μm; B, 5 μm; C (also applies to D–H), 0.5 μm.

Fig. 8.

Light and electron micrographs illustrating the innervation of dopaminergic neurons of the substantia nigra pars compacta (TH1–TH3; visualized with BDHC) by pallidal neuron 9678 (visualized with Ni-DAB) that also innervated interneurons in the neostriatum (Figs. 6, 7) and also projected to the EP, STN, and SNr (Figs. 1, 6, 7, 9). A, Low-magnification electron micrograph of a region of the substantia nigra pars compacta that contains the somata of three TH-IR neurons (TH2 is lightly labeled) and three pallidonigral boutons (b1–b3). B, C, Light micrographs of the same region shown in A at two different focal depths illustrating six boutons (b1–b6) apposed to the three TH-IR neurons. The two capillaries (c) act as further registration marks between the light and electron microscopes. D–I, High-magnification electron micrographs of the boutons (b1–b6) illustrating their synaptic contact with the three TH-IR neurons. Each bouton forms symmetrical synaptic contacts with the soma of the dopaminergic neurons (arrows). Note that individual boutons may possess multiple release sites (D, E, I). In the electron micrographs the amorphous nature of the Ni-DAB product (A, D–I) and the crystalline nature of the BDHC reaction product (visible in E, F) are apparent. Scale bars: A, B, 10 μm; D (also applies toE–I), 0.5 μm.

Fig. 9.

Light and electron micrographs illustrating the innervation of a PV-IR neuron of the substantia nigra pars reticulata by the axon terminals of pallidal neuron 9678 (visualized with NiDAB) that gave rise to the synaptic contacts in Figures 6, 7, and 8.A, Electron micrograph of a region of the substantia nigra pars reticulata that contains the somata of two PV-IR neurons (PV1, PV2; visualized with DAB) and two boutons (b1, b3) that appose PV1.B–D, Light micrographs of the same region shown inA at three different focal depths illustrating four boutons (b1–b4) apposed to PV1. A capillary (c) acts as a further registration mark of the light and electron micrographs. E–H, High-magnification electron micrographs of the boutons (b1–b4) illustrating their synaptic contacts with the soma of the PV-IR neuron PV1. Each bouton forms symmetrical synaptic contacts (arrows) with the soma of the GABAergic neuron. Note that individual boutons may possess multiple release sites (E, G). In the electron micrographs (A, E–H) the amorphous nature of the Ni-DAB reaction product is apparent. Scale bars: A, B (also applies to C, D), 10 μm; D (also applies to E–I), 0.5 μm.