Identification of Cholinergic Pallidocortical Neurons

Two recent studies identify and report the direct projections from the external globus pallidus (GPe) to the frontal cortex in rodents 1, 2. Some of the pallidocortical projections are identified from cholinergic neurons, and these cholinergic neurons respond to striatal stimulation 1, which raises a question on the distribution of cholinergic neurons in the GPe, and its relationship with other corticopetal cholinergic neurons in the basal forebrain. McKinney et al. and Saper first demonstrate that the basal forebrain contains corticopetal cholinergic and noncholinergic neurons of topographical nature 3, 4. Zaborszky and colleagues later further investigate topographical property in detail 5. It is generally thought that the corticopetal cholinergic neurons in the basal forebrain are homogeneous, cholinergic neurons in the GPe are probably misplaced.

The GPe at the rostral level contains cholinergic neurons that mostly locate in the (medial, ventral and lateral) borders or transition region between the GPe and basal forebrain. At the caudal level, due to lack of the landmarks for the GPe, it is difficult if not impossible to know whether the GPe contains cholinergic neurons. The ventral pallidum (VP) receives the ventral striatal inputs and anatomically forms a rostral and ventral extension of the GPe as the GPe is also called dorsal pallidum.

To delineate the pallidal cholinergic neurons, we crossed adenosine A2a receptor‐cre mice and mice with loxP flanked STOP cassette preventing tdTomato to obtain A2a‐cre‐tdTomato male mice (n = 4). Because A2a receptor and dopamine D2 receptor are co‐localized in the striatopallidal neurons in the striatum, and the axonal projections from A2a receptor neurons outline the GPe and VP, by labeling axonal terminals from the A2a neurons, we were able to identify and distinguish cholinergic neurons in the ventral VP and GPe and basal forebrain precisely.

Mapping striatopallidal terminal fields by mCherry labeling and choline acetyltransferase (ChAT, 1:1000, Chemcon, Temecula, CA, USA), we identified many cholinergic neurons in Уthe border/transition regionФ actually inside the GPe. We also identified cholinergic neurons in the VP (Figure 2). At the caudal level (Figure 2D,E), almost all the cholinergic neurons located in the GPe. By counting cholinergic neurons at five representative levels in the VP‐GPe and basal forebrain, we estimated that the number of cholinergic neurons in the VP and GPe was similar to the number of cholinergic neurons extra‐VP/GPe basal forebrain (total number added in five level in the second and third rows = total number added in five levels in the fourth row in Table 1).

Figure 2.

Distribution of cholinergic neurons in the VP and GPe. Cholinegric neurons labeled by ChAT (black color) are clearly seen within the VP and GPe marked by striatopallidal terminals of A2a neurons (brown color) from rostral to caudal level (A–E). In A, the VP (AP = 0.1 mm) marked by A2a neuronal terminals (brown color) contains cholinergic neurons (black color). The VP in the box is enlarged in a separate image (right). In B, GPe becomes prominent and contains cholinergic neurons mostly in its border regions. At further caudal level C–E, more and more cholinergic neurons are evenly distributed in the GPe. The cholinergic neurons in the striatum are interneurons projecting within the striatum.

Table 1.

Distribution of cholinergic neurons (number/side/section) in the VP and GPe and basal forebrain (n = 4) from rostral (AP = 0.1 mm) to caudal level (AP = −1.2 mm)

AP	0.10 mm	−0.40 mm	−0.6 mm	−0.9 mm	−1.20 mm
VP	21.7 ± 2.9	0	0	0	0
GPe	0	31.0 ± 5.8	26.2 ± 2.9	52.5 ± 6.4	48.7 ± 8.5
BF	49.7 ± 4.2	60.0 ± 9.1	49.5 ± 4.2	21.5 ± 3.1	2.5 ± 1.2

ChAT‐positive neurons are counted at five levels (AP: 0.1–1.2 mm) in the VP/GPe and basal forebrain (BF) per section per side. Roughly, number of cholinergic neurons in the VP/GPe (178 neurons) is equal to number of the cholinergic neurons (181 neurons) in the basal forebrain.

The GPe is defined by the projection field from dopamine D2‐adenosine A2a receptor containing neurons in the dorsal striatum (or caudoputamen, indirect pathway), while the VP is defined the projection field from D2/A2a receptor containing neurons in the ventral striatum (or accumbens) (Table 1, Figure 1). Because of lack of land markers for the GPe and VP, it has been difficult to ascertain if cholinergic neurons are in the GPe or VP. The basal forebrain corticopetal neurons have not been considered the part of the basal ganglia. Now we clearly show that cholinergic neurons equally distribute in the basal forebrain and VP‐GPe. In the rostral level, cholinergic neurons mostly distribute on the medial and ventral GPe, while at the caudal level, most cholinergic neurons evenly distribute within the GPe (Table 1, Figure 2). The percentage of cholinergic neurons in the GPe progressively increases along the rostrocaudal axis. It is possible that the corticopetal GABAergic neurons concentrate in the rostral GPe, while corticopetal cholinergic neurons concentrate in caudal GPe. In general, rostral GPe mainly projects to the frontal cortex while caudal GPe projects to sensory cortex (somatosensory, auditory, visual and posterior insular cortex) and rhinal cortex as well as the basolateral amygdala 6, 7.

Figure 1.

Projections of striatal A2a neurons outlining the GPe and VP. tdTomato labeled A2a receptor containing neurons in the striatum project to the GPe and VP. Because of heave converging terminals from the striatum, bright VP and GPe are very distinct from the surrounding regions. A–C shows the VP, GPe and basal forebrain from rostral to caudal level.

The GPe at rostral and caudal level differs in terms of projections. For instance, although caudal GPe projects to the caudal striatum, it does not project to other basal ganglia structures such as subthalamic nucleus (STN). Furthermore, the caudal GPe has a unique projection to the area dorsal to the dorsolateral SNr and cuneiform nucleus 8.

Based on retrograde tracing and distribution of cholinergic neurons, there are two clusters of cholinergic populations of corticopetal neurons 3. One population concentrates in the medial septum, diagonal band, magnocellular pre‐optic nucleus and the other one locates in the GPe and VP of the basal ganglia. Both populations project to the cerebral cortex, and the former one also projects to the hippocampus. Both cholinergic populations contain nerve growth factor receptor and neurotrophic tyrosine kinase receptor. Similarly, there are also separate corticopetal GABAergic populations of the basal forebrain and basal ganglia. Majority of GPe neurons fire faster at wake and REM (rapid eye movement) sleep than slow‐wave sleep, which is similar to the basal forebrain cholinergic neurons 9. The roles of cholinergic neurons in the GPe and VP in regulation of the cortex, motor and sleep–wake behavior, however, are not clear. Despite of similarity of firing pattern and morphology, given that the basal ganglia and basal forebrain have different afferents and functions, we hypothesize that cholinergic neurons in the GPe‐VP and basal forebrain have different influences on the cerebral cortex.

Identification of direct GABAergic and cholinergic pallidocortical projections has a significant implication in understanding the basal ganglia circuitry and regulation. It suggests that basal ganglia have an access to the cortex directly via the pallidocortical (GABAergic and cholinergic) projections. The pallidocortical pathway is the shortest in all possible anatomical routes from the basal ganglia to the cortex.

Conflict of Interest

The authors declare no conflict of interest.