Fiber connections of PoAb
Retrograde labeling by CTB. The center of the injection site of the right PoAb was placed at A 6.25, L 7.6, and D 6.8 according to the atlas of Pigeon13, and the diffusion of the tracer was mostly restricted within PoAb (Figure 1b). Labeled areas outside PoAb included many regions of the forebrain. Around PoAb, numerous labelings were found in the cortex piriformis (CPI) from approximately A 5.00 to A 7.50, fewer labelings were found in the arcopallium dorsale (AD) and arcopallium intermedium (AI), and sparse labelings were found in the arcopallium anterius (AA) (Figure 2c–f). Other labeled areas outside PoAb were divided into three streams in the telencephalon. Two of them were from the nidopallium caudoventrale, in which an extensively labeled area was found to be separated. One of them traveled dorsolaterally, along the way through the parahippocampalis (APH) and hippocampus (Hp) areas to the dorsolateral corticoid (CDL) and temporo–parieto–occipitalis (TPO) areas (Figure 2c–g); in contrast, the other ran ventrally and turned rostrally, passing through the nidopallium caudolaterale (NCL), dorsal mesopallium (MD), and ventral mesopallium (MV). Labeled neurons proceeding rostrally extended to the hyperpallium apicale (HA), hyperpallium densocellulare, hyperpallium ventrale (HV), and hyperpallium intercalatum (HI) of the rostral telencephalon (Figure 2a–c). They converged in the pallium at about A 7.0. The third course ran basal and turned rostrally. En route, it branched to nucleus taeniae amygdalae (TnA), nucleus septalis lateralis (SL) and the anterior commissura (AC). Labeled neurons proceeding further rostrally extended to the basorostral pallial nucleus.
Numerous labeled neurons were observed in the nucleus dorsomedialis anterior thalami (DMA) (Figure 2d–f) of the diencephalon. In contrast, fewer labeled neurons were observed in the radix mesencephalicus nervi trigemini (RxVM) (Figure 1e, Figure 2i), tectum opticum (TeO) (Figure 2e) and nucleus preopticus anterior (POA). Sparsely distributed labeled neurons were observed in the lateral hypothalamic nucleus (LHy) (Figure 2c) and nucleus rotundus.
In the midbrain, labeled neurons were widely scattered to a different site. CTB-labeled neurons were distributed in a continuous region stratum griseum central (SGC), stratum griseum et fibrosum superficiale (SGF) (Figure 1c–e, Figure 2g–i), nucleus isthmi, pars magnocellularis (Imc) (Figure 1c–d), nucleus isthmi, pars parvocellularis (Ipc) (Figure 1c–d), and formatio reticularis medialis mesencephali. Fewer labeled neurons were observed in the substantia grisea centralis (GCt) (Figure 2g–h), nucleus intercollicularis (ICo) (Figure 2g–h), substantia grisea et fibrosa periventricularis (SGP). Sparse labeling was observed in the locus ceruleus (LoC), nucleus pontis lateralis (PL), stratum album central (SAC) (Figure 1c–e, Figure 2h–i), nucleus subpretectalis (SP) (Figure 2f), and ventral tegmental area (VTA).
Anterograde labeling by BDA. The injection of BDA to the right PoAb was located at A 5.75, L 7.60, D 6.10, and diffusion of the tracer was restricted within the nucleus (Figure 1g). Similar to CTB-labeled neurons, around PoAb, many BDA-labeled fibers were found in CPI, AD, and AI. Moderate numbers of BDA-labeled fibers were observed in AA and arcopallium mediale (Figure 2c–f). In contrast, PoAc contained few labeled fibers. Rostral to the injection, projections from PoAb were divided into four streams: one traveled dorsolaterally of the forebrain, along the way through APH and Hp to CDL and TPO, and among them, CDL extensively contained BDA-labeled fibers (Figure 2c–g); in contrast, the other ran ventrally and turned rostrally, and the BDA-labeled fibers forming the continuum were found to exist extensively throughout NCL, MD, and MV. Labeled fibers extended to the HA, proceeding further rostrally. Less numerous labeled fibers existed in HV, and very sparsely labeled areas were observed in HI of the rostral telencephalon (Figure 2a–c). The first two streams joined in the pallium, similar to the findings observed for CTB labeling. The third stream ascending fiber bundle extended rostrally toward the basal forebrain, and on this pathway, many fibers appeared in TnA (Figure 2e). Moderate numbers of fibers appeared in nucleus septalis lateralis and tuberculum olfactorium, and more rostrally, terminated in AC at approximately A 7.75. Using AC as a turning point, the contralateral bundle repeated its ipsilateral in a mirror or symmetric fashion. The fourth stream ran through the diencephalon and midbrain via a descending fiber bundle.
In the diencephalon, the main course of the descending pathway was primarily through the following areas: DMA and DMP in the rostral diencephalon (Figure 2e–f); then TeO and POM in the caudal diencephalon; and finally, a limited number of fibers in nIV, LHy, and RxVM.
In the midbrain, the BDA-labeled areas were much more than the CTB-labeled areas. The descending pathway labeled many areas along its course, and the labeled areas were divided into two streams. Ventrally, it projected massively to the limbic midbrain. There were several labeled varicose fibers or terminals in fasciculus longitudinalis medialis, SGC, SGF, and SP (Figure 1h–j, Figure 2g–i). In addition, BDA-labeled neurons were frequently observed in GCt, SAC, SGP, and Sop (Figure 2g–i). The other labeled fibers were scattered in the midbrain, and these areas received a moderate number of labeled fibers from the descending pathway, including Imc, Ipc, OM and LoC (Figure 1j). Few labeled fibers were distributed sparsely in torus semicircularis, VTA, and tractus vestibule–mesencephalicus.
In the CTB and BDA injections, three pigeons each were were targeted to PoAb. In each case, two were largely confined to the PoAb and with successful labeling, so they were selected to describe and depict the results. And the summary Figures and Table 1 represent composites results. The general patterns of the afferent and efferent projections of PoAb are summarized in Figure 3 and Table 1.
Table 1
Distribution of labeling of BDA and CTB injection in PoAb
Sites
|
Number of CTB - labeled neurons
|
Density of BDA - labeled
fibers and terminals
|
Ipsilateral
|
Contralateral
|
Ipsilateral
|
Contralateral
|
Telencephalon
|
|
|
|
|
AA (arcopallium anterius)
|
+
|
-
|
++
|
-
|
AC (anterior commissura)
|
+++
|
+++
|
+++
|
+++
|
AD (arcopallium dorsale)
|
++
|
++
|
+++
|
+
|
AI (arcopallum intermedium)
|
++
|
++
|
+++
|
+
|
AM (arcopallium mediale)
|
-
|
-
|
++
|
-
|
APH (area parahippocampalis)
|
+++
|
+++
|
+++
|
+++
|
BaS (basorostral pallial nucleus)
|
+
|
+
|
-
|
-
|
CDL (area corticoidea dorsolateralis)
|
+++
|
+++
|
+++
|
+++
|
CPi (cortex piriformis)
|
+++
|
+++
|
+++
|
+++
|
HA (hyperpallium apicale)
|
++
|
+
|
+++
|
+++
|
HD (hyperpallium densocellulare)
|
++
|
+
|
-
|
-
|
HI (hyperpallium intercalatum)
|
+
|
-
|
++
|
-
|
Hp (hippocampus)
|
++
|
++
|
+++
|
+
|
HV (hyperpallium ventrale)
|
-
|
-
|
+
|
+
|
MD (mesopallium dorsale)
|
+++
|
+++
|
+++
|
+++
|
MV (mesopallium ventrale)
|
+++
|
+++
|
+++
|
+++
|
NCL (nidopallium caudolaterale)
|
+++
|
+++
|
+++
|
+++
|
NCV (nidopallium caudoventrale)
|
+++
|
+++
|
++
|
++
|
SL (nucleus septalis lateralis)
|
++
|
++
|
++
|
++
|
TnA (nucleus taeniae amygdalae)
|
+
|
+
|
+++
|
-
|
TPO (area temporo–parieto–occipitalis)
|
+++
|
-
|
+++
|
-
|
Tuo (tuberculum olfactorium)
|
-
|
-
|
++
|
++
|
Diencephalon
|
|
Distribution of Labeling of BDA and CTB Injection in PoAb (Table 1 continued)
|
DMA (nucleus dorsomedialis anterior thalami)
|
++
|
+++
|
+++
|
+++
|
DMP (nucleus dorsomedialis posterior thalami)
|
-
|
-
|
++
|
++
|
nIV (nucleus nervi trochlearis)
|
-
|
-
|
+
|
+
|
LHy (lateral hypothalamic nucleus)
|
+
|
+
|
+
|
+
|
POA (nucleus preopticus anterior)
|
++
|
++
|
-
|
-
|
POM (nucleus preopticus mediali)
|
-
|
-
|
++
|
++
|
Rt (nucleus rotundus)
|
+
|
+
|
-
|
-
|
RxVM (radix mesencephalicus nervi trigemini)
|
++
|
++
|
+
|
+
|
TeO (tectum opticum)
|
++
|
++
|
++
|
++
|
Midbrain
|
Distribution of Labeling of BDA and CTB Injection in PoAb (Table. 1 continued)
|
FLM (fasciculus longitudinalis medialis)
|
-
|
-
|
+++
|
+++
|
FRM (formatio reticularis medialis mesencephali)
|
+++
|
+++
|
-
|
-
|
GCt (substantia grisea centralis)
|
++
|
++
|
++
|
++
|
Ico (nucleus intercollicularis)
|
++
|
-
|
-
|
-
|
Imc (nucleus isthmi, pars magnocellularis)
|
+++
|
+++
|
++
|
++
|
Ipc (Nucleus isthmi, pars parvocellularis)
|
+++
|
+++
|
++
|
++
|
LoC (locus ceruleus)
|
+
|
+
|
++
|
++
|
OM (tractus occipito-mesencephalicus)
|
-
|
-
|
++
|
++
|
PL (nucleus pontis lateralis)
|
+
|
+
|
-
|
-
|
PT (nucleus pretectalis)
|
-
|
+
|
-
|
-
|
SAC (stratum album central)
|
+
|
+
|
++
|
++
|
SGC (stratum griseum central)
|
+++
|
+++
|
+++
|
+++
|
SGF (stratum griseum et fibrosum superficia)
|
+++
|
+++
|
+++
|
+++
|
SGP (substantia grisea et fibrosa periventricularis)
|
++
|
-
|
++
|
++
|
Sop (stratum opticum)
|
-
|
-
|
++
|
++
|
SP (nucleus subpretectalis)
|
+
|
+
|
+++
|
+++
|
SpL (nucleus spiriformis lateralis)
|
-
|
+
|
-
|
-
|
ToS (torus semicircularis)
|
-
|
-
|
+
|
+
|
TVM (tractus vestibule–mesencephalicus)
|
-
|
-
|
+
|
+
|
VTA (ventral tegmental area)
|
+
|
+
|
+
|
+
|
Number of labeled neurons and density of labeled fibers and terminals: +++, numerous; ++, moderate; +, few; -, absent.
Abbreviations: BDA, biotinylated dextran amine; CTB, cholera toxin subunit B; PoAb, basal division of posterior pallial amygdala
|
The effects of electrical stimulation on motor behaviors
Before the electrical stimulus, the pigeons stays in place, generally accompanied by turning its head, or raising its head or pecking feathers in the arena. A series of new different motor behaviors were induced after the electrical stimulation of PoA, among which ipsilateral lateral movement, forward movement, contralateral lateral movement, backward movement, etc., occurred more frequently (Figure 4a, 4d). The different motor behaviors elicited in the arena were defined using a previous study14 with slight modifications (see Table2 for details). Figure 4c showed that PoAb mainly mediated ipsilateral lateral movement (75.00%) and seldomly mediated contralateral lateral movement (1.85%). PoAc was more complex; the behavioral response of it was as follows: ipsilateral lateral movement (78.89%), contralateral lateral movement (3.89%), forward movement (1.11%), and backward movement (2.78%). The result indicated that PoA mainly mediated the turning movement behavior in pigeons. There was no significant difference in the ipsilateral lateral movement response rate between PoAc and PoAb (Figure 4f). The locations of the stimuli in PoAc and PoAb are presented in Figure 4b and Figure 4e, respectively.
Table 2
Definitions of some motor behaviors in the arena
Motor behaviors
|
Definition of the motor behaviors
|
Ipsilateral lateral movement
|
Alternate stepping of both feet towards right
side of the body
|
Forward movement
|
Alternate stepping of both feet and moving forward
|
Contralateral lateral movement
|
Alternate stepping of both feet towards left side of the body
|
Backward movement
|
Alternate stepping of both feet and moving backward
|
The number of steps generated by different behaviors is generally 2-3 steps.
|
The effects of different drugs on forward behavior
Personalized screening. According to the SMART output data, the total scores of 25 pigeons were obtained. A normality test was performed on the total scores of each pigeon, and the distribution was found to be non-normal (P > 0.05, N = 25, Figure 5a). Eight pigeons did not enter the central area (the total score was 0.00) (Figure 5b), and nine pigeons were very active in the central area (the total score was > 1.50, Figure 5d). Combining the locomotion trajectories of the pigeons in the experimental box captured by SMART, we finally selected those individuals of which the total scores between 0.90 and 1.50. Consequently, eight pigeons were used for the drug injection experiment (Figure 5c, 5e).
The effects of D1 and D2 receptor agonists and antagonists on the latency of entering central area in pigeons. The effects of D1 and D2 receptor agonists and antagonists on the latency period of pigeons entering the central area were different (Figure 5). D1 agonist decreased the latency period of pigeons for entering the central area (F3, 28 = 14.78, P < 0.001, Figure 5f). When the concentration of D1 agonist was 0.5 μg/μL, the range of the latency period of pigeons for entering the central area decreased by 44.61% (P < 0.001), but there was no significant difference in the other groups. In contrast, D1 antagonist increased the latency period of pigeons for entering the central area (F3, 28 = 27.58, P < 0.001, Figure 5g). When the concentration of D1 antagonist was 0.1 μg/μL and 0.5 μg/μL, the latency period increased significantly, and their ranges increased by 35.46% (P < 0.01) and 71.61% (P < 0.001), respectively. For 0.02 μg/μL concentration of D1 antagonist, the latency period did not increase significantly. D2 agonist also increased the latency period of pigeons for entering the central area (F3, 28 = 25.63, P < 0.001, Figure 5h). The ranges of the latency period increased by 45.24% (P < 0.001) and 81.40% (P < 0.001) when the concentrations of D2 agonist were 0.1 μg/μL and 0.5 μg/μL, respectively. However, 0.02 μg/μL D2 agonist exhibited no significant influence. D2 antagonist decreased the latency period of pigeons for entering the central area (F3, 28 = 7.74, P < 0.001, Figure 5i). The latency period of pigeons for entering the central area was decreased by 26.46% (P < 0.001) when the concentration of D2 antagonist was 0.02 μg/μL; but when the concentrations of D2 antagonist were 0.1 μg/μL and 0.5 μg/μL, the latency period was decreased by 16.69% (P < 0.05) and 18.89% (P < 0.05), respectively. Overall, D1 agonist and D2 antagonist can reduce the latency period of pigeons for entering the central area. In contrast, D1 antagonists and D2 agonists could increase the latency period of pigeons for entering the central area.
The effects of D1 and D2 receptor agonists and antagonists on the stay time of pigeon in the central area. D1 and D2 receptor agonists and antagonists have different effects on the stay time of pigeons in the central area (Figure 5). D1 agonist increased the stay time of pigeons in the central area (F3, 28 = 11.59, P < 0.001, Figure 5j). When the concentration of D1 agonist was 0.1 μg/μL and 0.5 μg/μL, the stay time of pigeons in the central area increased by 58.12% (P < 0.001) and 62.03% (P < 0.001), respectively, but when the concentration of D1 agonist was 0.02 μg/μL, no significant difference was noted in the stay time. D1 antagonist decreased the stay time of pigeons in the central area (F3, 28 = 26.19, P < 0.001, Figure 5k). The stay time of pigeons in the central area decreased by 40.21% (P < 0.001) when the concentration of D1 antagonist was 0.1 μg/μL. D2 agonist had a significant impact on the stay time of pigeons in the central area (F3, 28 = 34.64, P < 0.001, Figure 5l). When the concentration of D2 agonist was 0.02 μg/μL, the stay time in the central area increased by 53.72% (P < 0.001); whereas it decreased by 12.53% when the concentration of D2 agonist was 0.5 μg/μL, and no significant difference was noted. D2 antagonist increased the stay time of pigeons in the central area (F3, 28 = 81.74, P < 0.001, Figure 5m). The stay time of pigeons in the central area increased by 25.92% (P < 0.01) and 92.19% (P < 0.001) when the concentration of D2 antagonist was 0.1 μg/μL and 0.5 μg/μL, respectively; however, 0.02 μg/μL D2 antagonist did not result in significant changes in the stay time. In summary, with increase in drug concentration, D1 agonist and D2 antagonist significantly increased the stay time of pigeons in the central area. In contrast, D1 antagonist decreased the stay time of pigeons in the central area. Further, D2 agonist increased the stay time of pigeons in the central area at a low concentration but decreased the stay time at a high concentration.