Distribution, connection and function of ALDH1A1 + /TH + neurons in substantia nigra pars reticulata of mouse

The massive cell death of dopaminergic neurons (DANs) in substantia nigra pars compacta (SNC) is associated with motor diseases, such as Parkinson ’ s disease. Moreover, as a subtype of DANs in SNC, ALDH1A1 + neurons show better resistance to PD related neurotoxin. DANs can also be found in the substantia nigra pars reticulata (SNR), however, whether they are ALDH1A1 + neurons are rarely reported, as well as their projection, function, and reaction in the PD pathology. We studied the distribution of ALDH1A1 + neurons and track their projection by injecting pAAV. We figured out that, in SNR, 87 % neurons are ALDH1A1 + /TH + in ALDH1A1 + cluster averagely, while ALDH1A1 + /TH + : TH + is 52 % averagely. There are two enrichment regions of ALDH1A1 + /TH + neurons at brgma (cid:0) 3.40 mm and brgma (cid:0) 3.70 mm in the SNR of the nTg mice. Nevertheless, in one type of PD-liked mice model, the proportion of ALDH1A1 + /TH + : ALDH1A1 + neurons are 98 % averagely, while ALHD1A1 + /TH + : TH + is 57 %. Intriguingly, neuro-tracing discovered that there may be a previously unreported connection between SNR and anterior dorsal thalamus (ADT). The mouse received MPTP stereotactic injection to destroy TH + neurons in SNR showed depression behavior, indicated the DANs death in SNR may contribute to depression behavior.


Introduction
Aldehyde dehydrogenase 1 (ALDH1) plays an important role in cell proliferation, growth, and differentiation [11,44] , while ALDH1A1 is one of 19 ALDH1 isoforms, which are widely expressed in cancer stem cells, resisting chemotherapy, and resulting in high survival rate of cancer stem cells (Jie et al., 2018).There are only three nuclei that exhibit ALDH1A1 in the central nervous system: the substantia nigra, the red nucleus, and the ventral tegmental area [31].Most of ALDH1A1 + neurons are distributed in the ventral side of the TH + cell group in the substantia nigra pars compacta (SNC) [27].As of yet, no previous studies have examined the distribution of ALDH1A1 in substantia nigra pars reticulata (SNR).On the other hand, ALDH1A1 + neurons show better resistance to cytotoxic factors like α-Synuclein (α-syn), indicating that ALDH1A1 has a significant effect on neuron protection [27].Additionally, ALDH1A1 prevent cells death from cytotoxicity by promoting the detoxification of toxic molecules such as 3,4-dihydroxyphenylacetaldehyde (DOPAL) [27].To a certain extent, dopamine "leaked" into the cytoplasm during vesicular transport and metabolized by mitochondria into DOPAL.If DOPAL cannot be neutralized in time, it will lead to α-synuclein (α-syn named as following) deposit and cause PD [27].ALDH1A1, however, could prevents α-syn deposit by oxidizing DOPAL to 3,4-dihydroxyphenylacetic acid (DOPAC), a less destructive acid [13].
SNR neurons receive input from the outer cortex striatal macular region, while axons of ALDH1A1 + cells in the SNC output to dorsolateral striatum primarily [43].However, reports about ALDH1A1 + and TH + neurons in the SNR had been studied sparingly.There is a necessary to determine the distribution and function of ALDH1A1+/TH + neurons in SNR.Meanwhile, the connectivity between TH + neurons in SNR and other brain region remained unclearly.To answer these questions, we labeled and counted TH + neurons and ALDH1A1 + neurons to examine the rate of co-localization, and compared the number between the nTg mice and PD-liked mice i.e.Pixt3-tTA:Tet-O-A53T α-synuclein (PITX3/A53T named as following) model mice.We injected neuro-tracer (AAV) into to explore the SNR pathway.Moreover, we apply MPTP injection in the TH + neurons in SNR to study the potential function.

Animals and sampling
As describe previously, the heterozygous tetO-SNCA*A53T transgenic mice were used to crossbreed with the Pitx3+/IRES2-tTA knock-in transgenic mice to generate Pitx3/A53T mice [26].Non-transgenic (nTg) littermate mice served as controls.All experimental mice are 3month-old mice and kept on the C57BL/6J gene background.The mice were housed in a 12-h light/dark cycle in specific-pathogen-free conditions and fed a sterile regular diet ad libitum.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.
Before taking samples, the mice were paralyzed with pentobarbital at the dose of 0.03 ug/kg.The brains were taken and fixed for 48 h in 4 % Paraformaldehyde (Macklin).A solution of 20 % and 30 % sucrose in PBS solution was used to dehydrate it overnight after fixation.The temperature of the whole process kept at 4℃. Frozen tissue sections were cut by using a Leica CM1950 microtome with a 30um slice thickness.

Immunohistochemical DAB staining
Take one set out of every two sections from SNR.The primary antibody, Rabbit anti-TH antibody (1:1000, Pel-Freez), was incubated overnight at 4 degrees Celsius.Biotin goat-rabbit (1:500, Invitrogen) was incubated for one hour, following Vectastain Elite ABC Kit (Vector Laboratories) for 1 h.Color is developed on slices using the ImmPACT DAB peroxidase substrate (Vector Laboratories) for 3 min.Bake the secitons at 37 • C for 20 min and seal with sealing resin.

Nissl staining and cells counting
Brain tissue sections were stained in Cresyl Violet (Macklin) solution and dehydrated.Cell counting was perform by using the Axio Imager M2/MBF-SL/NL (n = 5, each experimental group).

Ethological experiment
A quiet and dark environment was secured for the ethological experiment after receiving MPTP stereotactic injections for 15 days (n = 10, each experimental group, male).
Rotarod test.The YLS-4C rod turning fatigue tester (Yiyan Technology Development Co., Ltd.) was used.Training was done with 10 rpm two days before the experiment, and 15 rpm the following day, four times a day with two-hour interval.The formal test started on the third day, and the time of falling was recorded [6].
Forced swimming test.A transparent beaker with 2 L of water is filled with 1 L of water each time.The mice were enclosed in as beaker filled with pure water containing 1 L.After 5 min, while they give up struggling, record the immobility time (floating state).The mice were dried under a warm lamp with towels after each test [34].
Tail suspension test.Mice's tails were hanged by a fixed iron frame and a clip.Tails are covered with straws to prevent the mice from creeping out.A non-dynamic trend was observed, in that mice attempted to escape but failed.The immobility time of mice within 8 min was recorded [41], Stukalin et al., 2020).
Morris water maze test.The Integrated Fine Behavior Monitoring and analysis system (Topscan, Clever Sys) and a round pool with a radius of 60 cm was used for video recording and mice tracking.Train the mice for five days conducted three times a day.On the 6th day, the platform was removed, and the activities were observed, including the retainment time in the quadrant where the platform was located and the times the mice swim crossed the platform area within one minute [30].
Spontaneous alternation experiment.A Y-shaped maze was used and three arms of the maze were labeled with different marks, and signed by 1, 2, and 3, all of which were 40 cm long.During each experiment, mice were placed in the same arm and recorded as they entered different arms within 10 min.Then we calculate the spontaneous alternation rate for each test: (spontaneous alternation times/ (selection times − 2)) * 100 % [19].

Statistics
SPSS Ver 25.0 was used for statistical analysis of data; interring groups by t-tests or one-way ANOVA.If p < 0.05, significant differences were considered.
H. Liu et al.

The distribution of TH + cells and ALDH1A1 + cells in SNR
TH + neurons distributed in a scattered arrangement along the medial part (Fig. 1.A-E) between brg − 2.80 mm and brg − 3.34 mm of SNR.According to the mouse brain atlas of Franklin's [33], there were two peak at brg − 3.34 mm (Fig. 1D, Fig. 2I) and brg − 3.79 mm (Fig. 1G, Fig. 2I).
Due to the lack of the knowledge about ALDH1A1 + cells in SNR, we costained ALDH1A1 and TH and showed the distribution of ALDH1A1 + cells and TH + cells in SNR is in most cases not significantly different (Fig. 2A-H).At the mid-dorsal area at brg − 3.43 mm section, however, was occupied by a large population of TH + single affirmative cells (Fig. 2E).ALDH1A1 + cells gather at the medial side did not co-staining with SNR TH + cells located on the dorsolateral side at brg − 3.79 mm (Fig. 2G).ALDH1A1 + neurons have two enrichment regions on the coronal plane, including the location in brg − 3.43 mm and brg − 3.79 mm (Fig. 2I).The tendency of the number of ALDH1A1 + and TH + neurons are similar.And the cell number of ALDH1A1+/TH + neurons is closed to ALDH1A1 + neurons, averagely 87 %, while the proportion of ALHD1A1+/TH+: TH+, averagely 52 %.ALDH1A1 + cells no longer exist at brg − 3.85 mm, meanwhile, the TH + cells are found inward (Fig. 2H).
The distribution of ALDH1A1 + cells in substantia nigra is different from that of TH + cells, which can be summarized as three characteristics.First, the ALDH1A1 + cell distribution is more concentrated than that of TH + cells.Secondly, ALDH1A1 + cells were mostly found in the medial portion, and less in the lateral and middle regions.Thirdly, ALDH1A1 + cells and TH + cells appeared simultaneously in the rostral substantia nigra, whereas ALDH1A1 + cells merged out in the caudal substantia nigra prior than TH + cells (Fig. 2J).

ALDH1A1+/TH + neurons decreased significantly in Pitx3/A53T mouse
Considering that extensive co-localization between TH + neurons and ALDH1A1 + neurons, we therefore calculated the number of TH + neurons and ALDH1A1 + neurons (Fig. 3A, C, D).Starting from the section of brg − 2.53 mm, we one set of the next two sections (interval 90 μm) and discovered that, as compared with the nTg mice, PA mice had significantly fewer ALDH1A1-/TH + cells (p = 0.015) and ALDH1A1+/TH + cells (p = 0.039), but the degree of reduction in ALDH1A1 + cells was less pronounced (Fig. 3B).The cell number of ALDH1A1 + neurons is closed to ALDH1A1+/TH + neurons, averagely 98 %, which is higher than nTg mice, while the proportion of ALHD1A1+/TH+: TH + is 57 % averagely.
These results may have suggested that ALDH1A1+/TH + neurons exhibit higher survival rate than that are ALDH1A1-/TH+.It consists with the former report that concern about the protective effect of ALDH1A1 in SNC, which means that ALDH1A1 may also play a protector for the DANs in SNR.

ADT is a target area for the projection TH + neurons in the SNR
For pursuing a further understanding the connectivity of TH + neurons in the SNR, downstream pathways were analyzed by using pAAV-TH-RFP-WPRE (Fig. 4A).Positive reaction can be observed in: Lateral 0.82 mm, anterior dorsal thalamus (ADT) (Fig. 4B) and Lateral 0.91 mm: large cell part/small cell part of the red nucleus (RMC/RPC) (Fig. 4C).The positive nerve projections in ADT indicating that it is possible that there is a neural pathway connecting SNR with ADT, which is realized through fiber bundles of TH + neurons.This pathway has never been reported before.With ADT being involved in learning and memory, depression, anxiety, and other emotional processes [37], it may be reasonable to assume that SNR is also involved.
To further confirm this connection, pAAV-TH-Cre-P2A-WPRE was injected into the AD region to retrogradely track the downstream TH + terminal with Cre protein (Fig. 4D).Next, AAV-Efla-DIO-mCherry-WPRE was injected into the SNR to activate the red fluorescence of Cre labeled cells specifically.The result shows a small number of cells in SNR triggered red fluorescent labeling.This is consistent with the fact that the number of TH + cells in SNR is small fundamentally.(Fig. 4E, F).
This pathway may provide us with a better understanding of the interaction between SNR and ADT regions, as well as related neurophysiological functions and diseases, such as spatial memory disorder, anxiety and depression [37].

Mice showed depression symptom after destroying TH + neurons in SNR with MPTP
Due to lack of data about the function of TH + neurons in SNR, we injected stereotaxic MPTP into the TH + neurons of SNR to assess whether ethological differences exist.
Compared with the mice injected with normal saline, the mice injected with MPTP had significantly less number of TH + neurons in the SNR (Fig. 5A) (p = 0.030), but the same situation did not exist in ALDH1A + neurons (Fig. 5B).In ethological tests we found that there was no significant difference between the MPTP group and the sham group in the rotarod test (Fig. 5E, F), we considered that the administration in SNR was not enough to cause the PD-liked motor.Intriguingly, in the tail suspension and forced swimming experiments, there was a significant difference between the two groups (tail suspension: p = 0.020; forced swimming: p = 0.038) (Fig. 5C, D).MPTP-injected mice refined more slowly in water and exhibit greater immobility in tail suspension than sham-operated mice, indicating that the injury of TH + neurons in SNR may cause depression related emotional disorders.Additionally, there was no significant difference in learning and memory in water maze (bouts: p = 0.794; Duration: p = 0.669) and spontaneous alternation tests (p = 0.653) (Fig. 5G, H, I).
The results of these experiments are only preliminary, but they still indicate that MPTP administration to SNR may cause depression behavior.

Discussion
The hallmark of Parkinson's disease is a substantial amount of the death of dopaminergic neuron in SNC.Furthermore, in SNC area, ALDH1A1 exhibits protective effects during neuropathy and resists pathological progress of PD [43].However, there were fewer reports in describing TH + and ALDH1A1 + neurons in SNR.
In our study, we discovered that TH + neurons were gathered in the dorsal medial part of SNR, as well as the caudal side (after brg − 3.20 mm).According to the previous report, in SNC, cells with ALDH1A1reaction would be TH- [43].Our results confirmed the same result in SNR.Moreover, the proportion of ALDH1A1+/TH+: TH + is 53 %, which indicated that only half of TH + neurons are ALDH1A1-, as well as in the PITX3/A53T mice.ALDH1A1 + neurons gathered on the medial side of SNR with but not dorsal side (Fig. 2J).The difference between ALDH1A1+/TH + neurons and ALDH1A1-/TH + neurons in SNR, which the meaning to the mDANs resistance in the SNR, remained to be answered in future research.
The deposition of α-Syn is one of the most characteristic PD symptoms which could cause the reduction of dopaminergic neuron.ALDH1A1, on the other hand, can protect cells from α-Syn toxicity and reduce apoptosis [27].In our study, the reduction degree of ALDH1A1+/TH + neurons in the PD model mice was less pronounced as that of ALDH1A1-/TH + neurons.These results may have suggested that ALDH1A1+/TH + neurons exhibited higher survival rate than that of the ALDH1A1-/TH + neurons, which indicated that ALDH1A1 may also play a protector for the DANs in SNR.Researchers had suggested that ALDH1A1 protects cells from cytotoxicity by neutralizing 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is a by-product of dopamine, when dopamine was continuously "leaked" during vesicular transport and oxidized by mitochondria in the cytoplasm into DOPAL [27,13].If DOPAL cannot be neutralized in time, it will lead to α-syn deposit by crosslinking.
One of the strongest dopaminergic projection was located in ADT.To further determine the converse pathway, we injected retrograde tracer and positive feedback was observed in the DANs of SNR.
As the documents revealed that ADT is related to diseases such as anxiety, depression, and spatial memory deficits, etc [37], there may derive a meaningful question: SNR possibly takes part in the diseases that related to ADT dysfunction.
To answer this question, we carry out preliminary study by stereotactic injection of MPTP to specifically generate dopaminergic neuron impairment in SNR.MPTP injected mice showed remarkable immobility in forced swimming test and tail suspension, which means that dopaminergic neuron in SNR may play a role in depression behavior.

Conclusion
This study revealed the neuroanatomical properties of ALDH1A1 + neurons and TH + neurons in SNR preliminarily.Two enrichment points are located on the caudal side, with a large amount of snout on the dorsomedial side.It may protect against neurodegenerative diseases by reducing the mortality of TH + neurons.Furthermore, our research unveiled that connectivity between ADT and SNR.The mouse with destroying TH + neurons in SNR showed depression behavior, indicated the DANs death in SNR may contribute to depression behavior.C) and (D), there is a significant difference on the immobility time of the forced swimming (p = 0.038) and mice of tail suspension (p = 0.020), which indicated the mice injected with MPTP may suffer from depression.(E) and (F),The rotarod test did not show significant differences (Falling times: p = 0.473; Sticking time: p = 0.794) between the two groups, indicated that there is no significant difference in motor behavior.(H) and (I) showed the events of the water maze test on the sixth day.There is no significant difference in Bouts and Duration (Bouts: p = 0.794; Duration: p = 0.669).In the Y-maze Spontaneous alternation test (G).The experiment showed that the learning and memory ability of the two groups of mice did not change significantly (p = 0.653).

Fig. 3 .
Fig. 3. TH + neuron number decreased in the SNR of Pitx3/A53T mice.(A) showed the distribution of of ALDH1A1 + and TH + cells in series of sections.There are two enrichment areas closed to brg − 3.34 mm and brg − 3.79 mm.(B) showed the statistics comparison of TH + neuron with or not ALDH1A1 + neuron in SNR between PITX3/A53T mice and nTg mice.(n = 3).Compared with nTg, the number of TH + neurons significantly decreased (mean of nTg mice: 151, mean of PITX3/ A53T mice: 71, p = 0.015); TH+/ALDH1A1 + cells in PA group also significantly decreased (mean of nTg mice: 73, mean of PITX3/A53T mice: 41, p = 0.039) cells.There was no significant change in the number of ALDH1A1 + cells (mean of nTg mice: 93, mean of PITX3/A53T mice: 40, p = 0.052).(C) and (D) showed the ALDH1A1+/TH + cells in brg-3.88mm of SNR of the nTg mice and PITX3/A53T mice, respectively.

Fig 0. 5 .
Fig 0.5.Reduction of TH + cell neuron number of the SNR maybe leaded to depression behavior.(A) There is a significant reduction of the TH + cell number of SNR after MPTP injection (mean of MPTP group: 7, mean of SHAM group: 11, p = 0.030) while no significance in ALDH1A1 + cells (mean of MPTP group: 7, mean of SHAM group: 8, p = 0.204) (B).(C) and (D), there is a significant difference on the immobility time of the forced swimming (p = 0.038) and mice of tail suspension (p = 0.020), which indicated the mice injected with MPTP may suffer from depression.(E) and (F),The rotarod test did not show significant differences (Falling times: p = 0.473; Sticking time: p = 0.794) between the two groups, indicated that there is no significant difference in motor behavior.(H) and (I) showed the events of the water maze test on the sixth day.There is no significant difference in Bouts and Duration (Bouts: p = 0.794; Duration: p = 0.669).In the Y-maze Spontaneous alternation test (G).The experiment showed that the learning and memory ability of the two groups of mice did not change significantly (p = 0.653).