Olfactory Bulb D2/D3 Receptor Availability after Intrastriatal Botulinum Neurotoxin-A Injection in a Unilateral 6-OHDA Rat Model of Parkinson’s Disease

Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson’s disease (PD) in humans. The first central relay of the olfactory pathway, the olfactory bulb (OB), depends, among other things, on an intact, functional crosstalk between dopaminergic interneurons and dopamine receptors (D2/D3R). In rats, hemiparkinsonism (hemi-PD) can be induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB), disrupting dopaminergic neurons of the substantia nigra pars compacta (SNpc). In a previous study, we showed that subsequent injection of botulinum neurotoxin-A (BoNT-A) into the striatum can reverse most of the pathological motor symptoms and normalize the D2/D3R availability. To determine whether this rat model is suitable to explain olfactory deficits that occur in humans with PD, we examined the availability of D2/D3R by longitudinal [18F]fallypride-PET/CT, the density of tyrosine hydroxylase immunoreactivity in the OB, olfactory performance by an orienting odor identification test adapted for rats, and a connectome analysis. PET/CT and immunohistochemical data remained largely unchanged after 6-OHDA lesion in experimental animals, suggesting that outcomes of the 6-OHDA hemi-PD rat model do not completely explain olfactory deficits in humans. However, after subsequent ipsilateral BoNT-A injection into the striatum, a significant 8.5% increase of the D2/D3R availability in the ipsilateral OB and concomitant improvement of olfactory performance were detectable. Based on tract-tracing meta-analysis, we speculate that this may be due to indirect connections between the striatum and the OB.

Since dopamine is a key transmitter in periglomerular inhibitory neurons of the OB [35][36][37][38], we speculate that hemi-PD may also lead to dopamine-associated alterations in the OB causing measurable olfactory impairment.
Positron emission tomography/computed tomography (PET/CT) using radioligand [ 18 F]fallypride enables the detection of the D 2 /D 3 receptor (D 2 /D 3 R) availability in vivo [56][57][58][59][60]. In a previous study, we applied [ 18 F]fallypride PET/CT to analyze the D 2 /D 3 R in the CPu of hemi-PD rats one, three, and six months after BoNT-A or Sham-BoNT-A injection [58]. There was a 23% increase of D 2 /D 3 R availability in the ipsilateral CPu of hemi-PD rats. Subsequently, intrastriatal injection of BoNT-A almost normalized the D 2 /D 3 R availability and reversed apomorphine-induced rotational behavior [58]. These results suggest a therapeutic effect of BoNT-A on the impaired motor behavior of hemi-PD rats by reducing interhemispheric differences of striatal D 2 /D 3 R.
We tested the hypothesis whether a unilateral 6-OHDA lesion of the right MFB is a suitable procedure to induce and study olfactory deficits in rats. We speculated that dopaminergic deafferentation of the CPu could lead to alterations in the expression of D 2 /D 3 R in the OB. Thus, in parallel approaches, D 2 /D 3 R availability was analyzed by dynamic [ 18 F]fallypride-PET/CT scans. Each rat was longitudinally scanned for OB D 2 /D 3 R availability 1, 3, and 6 months after intrastriatal BoNT-A or Sham-BoNT-A injection. In a subsequent approach using identical injection procedures, rats were tested for their olfactory abilities in the buried pellet test.
Additionally, pathway and graph distance analysis in combination with multivariate statistics were used to study the connectivity of the CPu and the OB, i.e., the olfactory-basal ganglia-connectivity (OBG), according to literature results on tract-tracing publications and its resulting possible planar network. 6-OHDA lesion of >95% was examined in apomorphine-induced rotational testing [61][62][63][64]. In the 6-OHDA animal model used here, dopaminergic deafferentation caused a compensatory increase in D 2 /D 3 R availability in the right CPu of hemi-PD rats by 23% [58]. This effect accounts for the motoric effect induced by injection of the D 2 /D 3 R agonist apomorphine [65][66][67][68].

Amphetamine-Induced Rotation Test
Amphetamine induces the release and inhibits the reuptake of dopamine in the striatum and causes ipsilateral rotation in animals with unilateral nigrostriatal lesions. In rats, amphetamine-induced rotation correlated both with the extent of the TH cell loss and with the degree of striatal dopamine deficiency [69][70][71][72][73].

Hemi-PD Rats Do Not Show Olfactory Deficits, but BoNT-A-Injected Hemi-PD Rats Improve Performance in the Buried Pellet Test
The buried pellet test, originally developed for mice, measures the food motivation aspect of olfaction, testing the ability of hungry (food restricted) animals to detect a palatable piece of sweetened cereal buried under bedding [74]. In this study, the test was adapted for rats for the first time.
Rats were tested daily on 5 consecutive days. As seen in Figure 1A, rats of neither group showed a learning effect throughout the experiments; the time (latencies) to find and start eating the pellet varied considerably between the days and the rats of the various groups. Means of latencies were calculated for each group ( Figure 1B). The latencies of non-injected (129.55 ± 17.56 s) and Sham + Sham (152.13 ± 20.95 s) rats did not differ significantly (p = 0.658). The respective values of 6-OHDA + Sham rats (127.02 ± 25.61 s) correspond roughly to those of the non-injected group. Thus, hemi-PD rats did not show significant olfactory deficits. Compared with Sham-treated hemi-PD rats (127.02 ± 25.61 s), 6-OHDA + BoNT rats (78.97 ± 13.47 s) tended to find the buried pellet faster (p = 0.253). However, the difference in latency between the 6-OHDA + BoNT rats and the Sham + Sham group was significant (p = 0.033) ( Figure 1B).
On testing day 6, the latencies were measured accordingly, but the pellet was now placed on the surface of the bedding (surface pellet test [74]) ( Figure 1C). In the surface pellet test, which was performed to exclude possible motor disorders or alterations in feeding motivation, the latencies to find the pellet did not differ between any of the four experimental groups ( Figure 1C). Median latencies were 7.00 s in the non-injected rats, 9.00 s in the Sham + Sham rats, 7.00 s in the 6-OHDA + Sham rats, and 7.50 s in the 6-OHDA + BoNT rats. Neither experimental group is likely to have impaired visual perception or spontaneous motor behavior in the given test situation. On testing day 6, the latencies were measured accordingly, but the pellet was now placed on the surface of the bedding (surface pellet test [74]) ( Figure 1C). In the surface pellet test, which was performed to exclude possible motor disorders or alterations in feeding motivation, the latencies to find the pellet did not differ between any of the four experimental groups ( Figure 1C). Median latencies were 7.00 s in the non-injected rats, Figure 1. Buried pellet test. (A) Latency to find the pellet in the buried pellet test of rats of the four groups during testing days 1 to 5. (B) Mean latency of rats of the four groups to find the pellet on days 1 to 5. Rats of the 6-OHDA + BoNT group were significantly faster in the buried pellet test than those of the Sham + Sham group. (C) In the surface pellet test, the latency to find the pellet was not significantly different among all four groups. Bar plots are given as means ± SD. Boxplots depict groups graphically by displaying the descriptive statistical parameters: median, upper, and lower quartiles, and outliers (circles) that lie outside the 10th and 90th percentiles (whiskers). Asterisks indicate significant differences after performing all multiple comparison procedures in pairs (Holm-Sidak or Dunn's method; * p < 0.05, ** p < 0.01).

There Is No Densitometric Interhemispheric Difference of TH-Immunoreactivity in the OB
The glomerular layer with its tyrosine hydroxylase (TH)-positive cells and nerve fibres, which is wrapped around the outer area of the OB similar to a band, can be seen clearly (Figure 2A-C). The stained specimens were scanned in 8-bit grayscale mode, and the perception or spontaneous motor behavior in the given test situation.

There Is No Densitometric Interhemispheric Difference of TH-Immunoreactivity in t
The glomerular layer with its tyrosine hydroxylase (TH)-positive cells an fibres, which is wrapped around the outer area of the OB similar to a band, can clearly (Figure 2A-C). The stained specimens were scanned in 8-bit grayscale mo the optical densities of the glomerular layer of both the left and right OB were dete in the 4 animal groups.  Densitometric analysis of the glomerular layer immunohistochemically reactive to TH of OB in both hemispheres revealed no significant interhemispheric differences in any of the 4 animal groups ( Figure 3A rats were 0.081 and 0.075, of the 6-OHDA + Sham rats were 0.075 and 0.074, and of the 6-OHDA + BoNT rats were 0.061 and 0.062. In addition, no significant differences between the optical densities of the glomerular layers were found between the respective values of the different experimental groups (p > 0.05) ( Figure 3A,B).
Toxins 2022, 14, x FOR PEER REVIEW 6 of 29 rats were 0.081 and 0.075, of the 6-OHDA + Sham rats were 0.075 and 0.074, and of the 6-OHDA + BoNT rats were 0.061 and 0.062. In addition, no significant differences between the optical densities of the glomerular layers were found between the respective values of the different experimental groups (p > 0.05) ( Figure 3A,B).
For quantification, the simplified reference tissue model 2 (SRTM2) was applied, and the non-displaceable Binding Potential (BPnd), which is used to measure the receptor transporter density [59], was estimated separately for the left and right OB in each experimental group at each time point. Remarkably, there were no significant differences between the absolute values of the BPnd of the Sham + Sham and the 6-OHDA + Sham rats (Table 1; Figure 4A,B,D,E,G,H,J,K). However, the right OB of BoNT-treated hemi-PD rats in PET/CT 2 exhibited significant results, about 8.5% higher BPnd values compared with both Sham + Sham and 6-OHDA + Sham rats (Table 1, Figure 4H). This may speak to BoNT-A-induced D2/D3R upregulation in the OB of hemi-PD rats after ipsilateral intrastriatal BoNT-A injection.
For quantification, the simplified reference tissue model 2 (SRTM2) was applied, and the non-displaceable Binding Potential (BP nd ), which is used to measure the receptor transporter density [59], was estimated separately for the left and right OB in each experimental group at each time point. Remarkably, there were no significant differences between the absolute values of the BP nd of the Sham + Sham and the 6-OHDA + Sham rats (Table 1; Figure 4A,B,D,E,G,H,J,K). However, the right OB of BoNT-treated hemi-PD rats in PET/CT 2 exhibited significant results, about 8.5% higher BP nd values compared with both Sham + Sham and 6-OHDA + Sham rats (Table 1, Figure 4H). This may speak to BoNT-A-induced D 2 /D 3 R upregulation in the OB of hemi-PD rats after ipsilateral intrastriatal BoNT-A injection.
Calculation of relative interhemispheric differences of BP nd values revealed more differentiated results. In controls (Sham + Sham), no significant relative interhemispheric right/left differences were found: In PET/CT 1, mean BP nd values of the left OB (2.93 ± 0.26) and the right OB (2.96 ± 0.29) showed a small, non-significant relative interhemispheric difference of 1.32 ± 1.66% (Table 1, Figure 4C,F). Respectively, relative interhemispheric difference in the same range were found in PET/CT 2 and PET/CT 3 (Table 1, Figure 4C,I,L). Table 1. Overview of rat-specific single BP nd values of the D 2 /D 3 R for the left and right OB, as well as the interhemispheric difference relative to the left hemisphere, in [%] in all rats analyzed. Data are shown for three PET/CT scans of the longitudinal study design (PET/CT 1: one month post-BoNT-A or Sham-BoNT-A, PET/CT 2: three months post-BoNT-A or Sham-BoNT-A, PET/CT 3: six months post-BoNT-A or Sham-BoNT-A). n/a indicates that no data were analyzed due to incorrect tracer injection or no data acquisition. n/a n/a n/a n/a n/a n/a 6-OHDA + Sham 3.45 3.73 8.31 n/a n/a n/a n/a n/a n/a  Bar plots are given as means ± SD. Boxplots graphically depict groups by displaying the descriptive statistical parameters: median, upper, and lower quartiles, and outliers (circles) that lie outside the 10th and 90th percentiles (whiskers). Asterisks indicate significant differences after performing all multiple comparison procedures in pairs (Holm-Sidak or Dunn's method; * p < 0.05, ** p < 0.01, *** p < 0.001).

Connectomics-There Are No Direct Pathways between the Main Olfactory Bulb and Basal Ganglia
Based on the neuroanatomical components of the basal ganglia complex, as well as the main olfactory bulb (MOB), we first tested whether a direct neuronal connection exists between the MOB and CPu. This was found neither ipsilaterally nor contralaterally. Using the pathway analysis function of neuroVIISAS, all shortest pathways across intermediate regions between the CPu and the MOB were determined, and then filtered with respect to their geometric distance, as well as connection weights: The regions involved in Figure 5A,B represent the strongest connectivity between the MOB and CPu. The results were represented first as an adjacency matrix ( Figure 5A). The tenses of the adjacency matrix contain the same regions and the same region sequences as the columns, so it is a square matrix. The rows indicate the source regions of the neural connections, and the columns indicate the regions that receive the connections (target regions) ( Figure 5A). The connection weights represent semi-quantitative values in analogy to the data in the original research literature. Second, data are represented as a planar weighted and rectified orthogonal graph ( Figure 5B). There is no direct connection from the CPu to the MOB. All connections from CPu to MOB have at least one intermittent region. With regard to the frequency of observations in tract-tracing publications, there are three possibly modified pathways: CPu→dorsal raphe nucleus→MOB, CPu→ventral tegmental area→MOB, CPu →amygdala complex→MOB are the most promising candidates for the BoNT-A-induced D 2 /D 3 R increase in hemi-PD rats ( Figure 5B).

Correlating Buried Pellet Test, Apomorphine-and Amphetamine-Induced Rotations, and Optical Densites of Glomerular Layers of the Left and Right OB
Prior to the buried pellet test, all animals were subjected to apomorphine-and amphetamine-induced rotation tests approximately two weeks after injection of the vehicle or BoNT-A ( Figure 6A-D). With the correlation analysis, we checked whether there was a dependence of parameters measured in the buried pellet test on apomorphine-and amphetamine-induced rotations. If so, the results of the drug-induced rotations could possibly predict measures to indicate olfactory deficits in hemi-PD rats. In addition, we analyzed the effect of a unilateral intrastriatal BoNT-A injection in hemi-PD rats ( Figure 6B,D).
original research literature. Second, data are represented as a planar weighted and rectified orthogonal graph ( Figure 5B). There is no direct connection from the CPu to the MOB. All connections from CPu to MOB have at least one intermittent region. With regard to the frequency of observations in tract-tracing publications, there are three possibly modified pathways: CPu→dorsal raphe nucleus→MOB, CPu→ventral tegmental area→MOB, CPu →amygdala complex→MOB are the most promising candidates for the BoNT-A-induced D2/D3R increase in hemi-PD rats ( Figure 5B). The buried pellet test showed that the latency to find the pellet was significantly correlated with apomorphine-induced rotations in rats in the 6-OHDA + BoNT group: rats with shorter latencies had fewer clockwise rotations (rs = 0.709; p = 0.001; Figure 6B). The correlation is even significant if the two outliers with the lowest and highest rotation values are excluded from the analysis (rs = 0.629; p = 0.0087).
The latency to find the pellet of rats in the 6-OHDA + BoNT, 6-OHDA + Sham, and Sham + Sham groups after 6-OHDA lesion, and also after BoNT-A or vehicle injection, did not correlate with the optical densities of the glomerular layers of the left and right OB (Supplementary Materials, Figure S1A-D).
No significant correlations were found between the optical densities of the glomerular layers of the left OB with appropriate apomorphine-and amphetamine-induced rotations in 6-OHDA + Sham, 6-OHDA + BoNT, and Sham + Sham groups (Supplementary Materials, Figure S2A-D).
Correspondingly, also the optical densities of glomerular layers of the right OB in the 6-OHDA + Sham, 6-OHDA + BoNT, and Sham + Sham groups did not significantly correlate with respective apomorphine-and amphetamine-induced rotations (Supplementary Materials, Figure S3A-D).
vehicle or BoNT-A ( Figure 6A-D). With the correlation analysis, we checked whether there was a dependence of parameters measured in the buried pellet test on apomorphineand amphetamine-induced rotations. If so, the results of the drug-induced rotations could possibly predict measures to indicate olfactory deficits in hemi-PD rats. In addition, we analyzed the effect of a unilateral intrastriatal BoNT-A injection in hemi-PD rats ( Figure  6B,D).

Discussion
Here, we present a longitudinal study of changes in olfactory bulb D 2 /D 3 R availability in the 6-OHDA-induced hemi-PD rat model and results of an olfactory detection test.
Hemi-PD rats did not show significant olfactory deficits in the buried pellet test. However, 6-OHDA + BoNT rats found the hidden pellet significantly faster than animals of the control group. Densitometric analysis of TH-immunoreactivity in the glomerular layer of the OBs of both hemispheres showed no significant interhemispheric differences in any of the 4 animal groups. When right and left D 2 /D 3 R availability in the OBs were measured, the right OB of BoNT-A-treated hemi-PD rats 6 weeks, 3 months, and 6 months (PET/CT 1-3) after BoNT-A injection showed significant, about 8.5% higher, BP nd values in the right OB compared with both Sham + Sham and 6-OHDA + Sham rats. Apparently, BoNT-A injected intrastriatally induced D 2 /D 3 R upregulation in the OB of hemi-PD rats.
Consistent with the hypothesis that the CPu might affect the OB, resulting in olfactory deficits, a tract-tracing meta-study revealed no direct neuronal connection, based on more than one publication, between the OB and CPu in rats. However, promising candidates for a D 2 /D 3 R regulation between the CPu and the OB are intermittent pathways via the dorsal raphe nucleus or the ventral tegmental area or the amygdala complex. Testing for correlations between the parameters examined in this study revealed that the latency to find the buried pellet was strongly correlated with apomorphine-induced rotations of rats in the 6-OHDA + BoNT group: rats with shorter latencies had fewer anti-clockwise rotations. This indicates that striatal injection of BoNT-A improves olfactory performance.

Hemi-PD Rats Show No Olfactory Deficits in the Buried Pellet Test
Although a few studies have described the 6-OHDA animal model with unilateral or bilateral lesion of the SN or the OB, or both [107][108][109][110][111][112][113], no olfactory behavior tests have been performed. A few studies using these tests have been done, however, only in differently designed 6-OHDA or MPTP mouse models [76,101,104,114,115] or in mice with neurodegenerative diseases, such as Niemann-Pick Disease Type C1 [116]. Outcomes of our buried pellet test demonstrated that rats of neither group showed olfactory deficits. Remarkably, intrastriatal BoNT-A injection in hemi-PD rats significantly improved olfactory performance in the buried pellet test in 6-OHDA + BoNT rats compared with the Sham + Sham group. Compared with the Sham-lesioned hemi-PD rats, 6-OHDA + BoNT rats tended to find the buried pellet faster (but not significantly). This correlated well with our results obtained from PET/CT experiments on D 2 /D 3 R availability in the OBs of the same animal group and highlights the importance of the dopaminergic balance in the OB.
Taken together, hemi-PD rats showed no olfactory deficits. However, intrastriatal BoNT-A injection in hemi-PD rats significantly improved latency in the buried pellet test in 6-OHDA + BoNT rats compared with the Sham + Sham group and, tendentially, also compared with the 6-OHDA + Sham rats.

Dopamine as a Key Transmitter in Processing of Olfactory Information
In order to understand functional changes in the OB after unilateral lesion of the MFB, we studied the behavior of both dopaminergic interneurons by immunohistochemistry and the correspondent availability of D 2 /D 3 R using PET/CT. Dopamine is a key transmitter for processing olfactory information in the glomerular layer of the OB, which is a crosstalk between dopaminergic interneurons and D 2 receptors in the terminals of axons of olfactory and in presynaptic elements of the glomerular neuropil [117]. Thus, dopamine could act in glomerular circuits through presynaptic mechanisms mediated by D 2 receptors. Correspondingly, a high density of D 2 receptors was anatomically localized especially in the glomerular layer [118][119][120][121][122][123][124][125]. All structures related to olfaction show high numbers of D 2 and D 1 receptors [126].

Hemi-PD Rats Do Not Show Altered TH-Immunoreactivity in the Glomerular Layer of the OB
Synuclein-immunopositive Lewy bodies and Lewy neurites in the OB have been reported in very early stages of PD [24]. An increased number of olfactory dopaminergic glomerular cells in PD was first reported by Huisman and colleagues [127]. Since glomerular dopaminergic TH-immunoreactive interneurons in OB release dopamine and GABA, which would inhibit glutamatergic neurotransmission from receptor neurons to mitral cells [37,128,129], it has been suggested that the increased number of inhibitory TH-immunoreactive interneurons might cause hyposmia in PD [127,130]. In addition, the increase of dopamine levels in the OB possibly explains why, in PD, olfaction does not improve with levodopa therapy [131]. The increase in the number of dopaminergic neurons in the OB could reflect a compensatory mechanism, created by the early degeneration of other neurotransmitter systems, and might contribute to the olfactory dysfunction in PD patients. The hypothesis arose that this modification could be a general characteristic for other neurodegenerative disorders involving cholinergic, noradrenergic and serotonergic denervation. Cholinergic [132,133] and noradrenergic [134,135] centrifugal effects on the OB exert an inhibitory effect on the mitral cell layer of OB, while serotonergic input can activate periglomerular neurons to release GABA [136]. Therefore, a dysfunction of the centrifugal sense of smell input from an early degeneration of these systems could cause an inhibitory imbalance that could be corrected by increasing the number of THimmunoreactive periglomerular neurons. In order to evaluate whether lesioning of the SNpc via MFB injection of 6-OHDA is followed by changes in dopaminergic parameters in the rat OB, densitometric analysis of the glomerular layer of the OB, stained immunohistochemically against TH, was carried out in addition to the availability of the D 2 /D 3 R. Densitometric analysis in both hemispheres in our study revealed no significant interhemispheric differences in any of the 4 animal groups. In addition, no significant differences were found between the optical densities of the glomerular layers between the respective values of the different experimental groups. Thus, our study adds a further result to the conflicting reports on the TH-immunoreactivity in the OB of PD patients or rodent PD models. There are only two studies whose results demonstrated increased numbers of THimmunoreactive neurons after unilateral lesion trials in rats. Winner et al. [96] described changes 6 weeks after unilateral lesion of the MFB, and Voronkov et al. [110] reported an increase after unilateral SN lesion. The latter authors interpreted this as a possible compensatory process, and argued that a more pronounced dopamine-related inhibitory effect in the glomeruli of the OB could contribute to the development of hyposmia in PD. Olfactory behavior tests were not performed. An increased number of olfactory dopaminergic glomerular cells in the autoptic material of PD patients was first reported by Huisman et al. [127,130] and later observed in Alzheimer's disease and frontotemporal dementia patients when compared with age-matched controls [137]. Our results are supported by Ilkiw et al. [112], who found no increase in TH-immunoreactive periglomerular cells compared with controls, and, interestingly, also no changed measurements in the olfactory discrimination task after 6-OHDA lesion of the SN. Controversial results on the effect of unilateral lesion have also been reported in mice. Zhang et al. [76] observed no difference in the number of dopaminergic interneurons in the OB after injection in the SNpc, whereas Sui et al. [138], and Chiu et al. [139] reported a significant increase.

Intrastriatal BoNT-A Injection Led to an Upregulation of D 2 /D 3 Receptor Availability in the Ipsilateral OB of Hemi-PD Rats
We further analyzed the availability of the D 2 /D 3 R of the OB after unilateral 6-OHDA lesion into the right MFB, followed by unilateral intrastriatal BoNT-A or vehicle injection in male Wistar rats using [ 18 F]fallypride-PET/CT scans. We focused on olfaction-related parameters in hemi-PD rats with unilateral lesion of the SNpc and subsequent intrastriatal BoNT-A injection. Based on previously reported results on [ 18 F]fallypride-PET/CT in the CPu [58], we speculated that dopaminergic deafferentation induced by unilateral injection of 6-OHDA into the right MFB of the CPu via destruction of the SN may also lead to alterations in the expression of dopamine D 2 /D 3 R in the OB. Moreover, altered proliferation of adult neuronal progenitor cells in the subventricular zone and their migration to and positioning and differentiation in various neuron types in the OB seen in hemi-PD mouse models could result in changes in D 2 /D 3 R [138,139]. Our study also revealed that the induction of hemi-PD following 6-OHDA injection into the MFB did not alter the D 2 /D 3 R availability of the OBs. Surprisingly, although studied in other brain regions, data on the quantification of D 2 /D 3 R in the OB are not evaluable in PD patients [140,141] nor in animal models of PD [142,143].
The intrastriatal BoNT-A injection in hemi-PD rats, however, resulted in an 8.5% increase of the D 2 /D 3 R availability of the ipsilateral OB and is accompanied by a better olfactory test performance. The reasons for this, however, remain unclear.
The only significant correlation in the various parameters measured in the present study were found in the 6-OHDA + BoNT rats between the latency to find the buried pellets and apomorphine-induced rotations: the faster the pellets were found by the rats, the fewer rotations they made. The beneficial effect of intrastriatal BoNT-A in hemi-PD rats with respect to the reduction of apomorphine-induced rotational behavior could be explained by modifications in basal ganglia circuitry and changes in densities of dopaminergic and cholinergic receptors in the CPu [43,[45][46][47]52,53,58,144,145]. Rats of the 6-OHDA + Sham and Sham + Sham groups lacked any significant dependence of latency and apomorphineor amphetamine-induced rotations. Therefore, in hemi-PD rats, analysis of motor deficits is apparently not predicative for changes of olfaction performance.
The significantly increased interhemispheric difference of approximately 8% in D 2 /D 3 receptor availability in the ipsilateral OB of BoNT-A-treated hemi-PD rats is of biological relevance. Even, at first glance, small but significant differences in receptor density have been shown to be functionally significant. For example, in a study on circling rats, a significant difference in adenosine A1 receptor binding site density was found between ci2 rat mutants and wild types in the range of 10% in motor areas [146]. In wild type rats (LEW/Ztm strain), the CPu in the left hemisphere had a significantly (5%) lower density of kainate receptor binding sites than in the right hemisphere [147]. In aged Fischer 344/Brown Norway rats that differed in retention performance in a water maze reference memory task, superior and inferior learners had significantly different binding site densities of various receptors in the range of 10-15% [148]. In different mouse models with PD-associated gene mutations, quantitative multi-receptor studies revealed significant differences in receptor-binding sites in various brain areas, ranging from 9% to 42% [142,143].

Connectomics Reveal Indirect Projections between the OB and the CPu
The results of the PET/CT study may account for a compensatory D 2 /D 3 R increase in hemi-PD rats which were injected with BoNT-A into the dopamine-deprived CPu and could be explained most probably by a functional connectivity of the CPu and the OB. To clarify possible connectome pathways, olfactory connectome analysis provided evidence that both direct and indirect axonal connections may act as the structural backbone for regulating regional receptor expressions. We found that the glomerular layer of the OB had unilateral and reciprocal interconnectivity with the VTA, and the projection from OB to CPu is nonreciprocal. VTA is reciprocally connected with SN and CPu. This indirect connectivity of the OB may be the reason for D 2 /D 3 R upregulation after MFB lesion with subsequent intrastriatal BoNT-A injection. According to the hypothesis that the CPu might affect the OB and, thereby, possibly cause olfactory deficits, we found no direct neuronal connection between the OB and CPu in the literature. However, promising candidates for a D 2 /D 3 R regulation between the CPu and the OB are intermittent pathways via the dorsal raphe nucleus, the ventral tegmental area or the amygdala complex. In addition, BoNT-A may be transported in anterograde and retrograde directions and also transsynaptically [149,150], as demonstrated in the visual system of mice [151,152] and in the spinal cord of rats, as central effects of intramuscularly administered BoNT [153].
However, the molecular basis of the positive effect of intrastriatal BoNT-A application on D 2 /D 3 R density in the ipsilateral olfactory bulb has not been fully elucidated. Given the indirect morphologic connection between CPu and OB, the interhemispheric difference in D 2 /D 3 R density in the OB appears to be unexpectedly high compared with a difference of approximately 25% within the CPu [55]. We hypothesize that an interhemispheric difference in D 2 /D 3 R availability would have been even more pronounced if the OB had been injected directly.

Animals
Fifty-one young adult, 3 month-old, male Wistar rats (strain Crl:WI BR, Charles River Wiga, Sulzfeld, Germany) weighing 295-310 g at the time of the first surgery were used. Animals were housed in standard cages in a temperature-controlled room (22 • C ± 2 • C) under a fixed 12 h light/dark cycle and had free access to food and water ad libitum. All procedures used complied with the guidelines on animal care. All experiments (Figure 7

Animals
Fifty-one young adult, 3 month-old, male Wistar rats (strain Crl:WI BR, Charles River Wiga, Sulzfeld, Germany) weighing 295-310 g at the time of the first surgery were used. Animals were housed in standard cages in a temperature-controlled room (22 °C ± 2 °C) under a fixed 12 h light/dark cycle and had free access to food and water ad libitum. All procedures used complied with the guidelines on animal care. All experiments (Figure 7

Induction of Hemiparkinsonism
All surgeries were carried out under aseptic conditions and deep anesthesia by intraperitoneal injection of a mixture of ketamine (50 mg/kg −1 BW) and xylazine (4 mg/kg

Injection of BoNT-A into the Striatum
Hemi-PD rats underwent a second stereotactic surgery six weeks after the 6-OHDA lesion and were treated with either BoNT-A (n = 18) or vehicle (n = 10). 2 × 1 µL BoNT-A solution (Lot No. 13028A1A; List, Campbell, CA, USA, purchased via Quadratech, Surrey, UK; BoNT-A dissolved in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA)) was injected with a total dose of 1 ng BoNT-A into the right CPu at two sites [43-46,48-50,52-54,144]. Sham-BoNT-A rats received 2 × 1 µL PBS + 0.1% Figure 7. Timeline of the study design. Hemi-PD was induced by 6-OHDA injection into the right MFB. Controls received a Sham-6-OHDA injection. The degree of dopaminergic cell loss was verified with apomorphine-and amphetamine-induced behavioral testing. Six weeks after the 6-OHDA or Sham-6-OHDA injection, rats obtained BoNT-A or Sham-BoNT-A injection into the ipsilateral CPu. The positive effect on the motor behavior of BoNT-A was then controlled in rotation tests. To verify alterations of the olfactory ability, as well as the effect of the intrastriatal BoNT-A injections on the olfactory function in hemi-PD rats, the buried pellet test was conducted. Subsequently, each rat was scanned by [ 18 F]fallypride-PET/CT analysis one, three, and six months post-BoNT-A or post-Sham-BoNT-A injection. A final MRI scan was performed as an anatomical reference for PET/CT imaging.

Induction of Hemiparkinsonism
All surgeries were carried out under aseptic conditions and deep anesthesia by intraperitoneal injection of a mixture of ketamine (50 mg/kg −1 BW) and xylazine (4 mg/kg −1 BW). To induce a unilateral degeneration of dopaminergic neurons in the SNpc and hemiparkinsonian symptoms, a unilateral injection of 24 µg 6-OHDA (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 4 µL of 0.1 M citrate buffer was performed over 4 min via a 26 gauge 5 µL Hamilton syringe into the right MFB using a David Kopf stereotactic frame. The Sham hemi-PD rats received 4 µL of the 0.1 M citrate buffer. Thereafter, the needle was left in place for another 5 min to avoid reflux. The injection coordinates with reference to bregma were: AP = −2.3, L = 1.5 to the right, V = −9.0 [154].
To verify alterations of the olfactory ability, as well as the effect of the intrastriatal BoNT-A injections on the olfactory function in hemi-PD rats, the buried pellet test according to Lehmkuhl et al. [74] as adapted to rats was conducted.
Before testing, animals were food-restricted for 3 days (food was available for 1 h per day) and thereafter maintained at about 90% of free-feeding body weight during testing procedures [157]. One week prior and during food restriction, each tested rat was accustomed to a piece of sweetened cereal pellet later to be buried (Honey Bsss Loops, Kellogg, Munich, Germany) and, therefore, received 2 pieces of the pellets every day.
On all testing days, rats were habituated to the testing room and, 1 h before the test, were kept in their home cage without a water bottle. For the first 5 testing days (buried pellet test), freshly cleaned testing cages (Makrolonbox typ IV, UNO BV, Zevenaar, Netherlands) were prepared with~3 cm clean bedding, and one pellet was buried 0.5 cm below in one corner of the cage. Importantly, every day the pellet was buried in a different spot in the cage for each trial, and the testing cage and experimenter's gloves were changed after each animal. For testing, each rat was removed from its home cage and placed in the center of the test cage; the latency time was measured until the rat uncovered the pellet and began eating it. If a rat did not find the pellet within the predetermined time of 300 s, the experiment was terminated, and a latency of 5 min was recorded. Additionally, the experimenter removed the pellet from the bedding, and the rat was allowed to eat it.
On testing day 6, the test was repeated using the same scheme, but now the pellet was placed on the surface (surface pellet test); the time when the rat found and started eating the pellet was recorded.
All trials were videotaped, and the latencies on testing days 1-5 (buried pellet test) and on testing day 6 (surface pellet test) were measured and expressed as means ± SEM.

TH-Immunohistochemistry of the Olfactory Bulb
Rats were killed with an overdose of ketamine/xylazine, and then the body circuit was perfused transcardially with 50 mL of 4 • C cold 0.9% saline, followed by 400 mL of 4% paraformaldehyde (in phosphate-buffered saline (PBS), 0.1 M, pH 7.4). The brains were immediately removed from the skull and postfixed overnight at 4 • C in 4% paraformaldehyde solution. Subsequently, brains were cryoprotected at 4 • C for 48 h in 20% sucrose solution and snap frozen in −50 • C cold isopentane. The brains were stored at −80 • C. Frontal 30 µm-thick brain slices were serially cut with a cryostat (Leica, Germany). Histological sections of the OB were immunohistochemically reacted with an antibody against tyrosine hydroxylase (TH). For this purpose, the sections were washed in PBS, endogenous peroxidases were blocked using 3% hydrogen peroxide solution, and non-specific binding sites were blocked using horse serum. Primary antibody incubation (monoclonal, anti-TH, mouse, 1:1000, Sigma-Aldrich, St. Louis, MO, USA) was performed overnight at 4 • C. Secondary antibody incubation (polyclonal, anti-mouse, horse, 1:200, Vector Laboratories, Burlingame, CA, USA) was also performed at 4 • C overnight. Immunohistochemical labeling was visualized using a standardized 3,3 diaminobenzidine hydrochloride (10 mg/100 mL phosphate-buffered saline, Sigma-Aldrich, St. Louis, MO, USA) procedure.

Densitometric Measurement
Measurements were performed in the glomerular layers of the left and right OB of the animals. Rats of the non-injected group (n = 3), the Sham + Sham group (n = 5), the 6-OHDA + Sham group (n = 5), and the 6-OHDA + BoNT group (n = 5) were evaluated. Frontal sections of the OB were scanned with a high-resolution scanner (Nexscan F4100, Heidelberger, Germany) using the transmitted light method with a resolution of 2650 dpi. Digital 8-bit grey value images were generated for further computer-aided evaluation. In the images, the glomerular layer was manually delineated using the program Icy 2.0.1.0 (BioImage Analysis unit Institut Pasteur Unite d'analyse d'images quantitative, Paris, France) to determine the grey values of the glomerular layer and the non-specific background. Data were exported to Excel ® and used to calculate the optical density. The negative decadic logarithm of the quotient from the grey values of the glomerular layer and the respective non-specific background resulted in the respective optical densities.

Atlas Generation and PET/CT Data Analysis
The D2/D3R availability was quantified with the sof Technologies LLC, Zurich, Switzerland). A reference mod displaceable Binding Potential (BPnd) was determined. For thi (left and right OB) were delineated in a first step using a self-d atlas. For the generation of the atlas, the OB was defined in different experimental groups in each slice of the MRI datase (Schiffer space [158]) fitting the anatomical dimensions of PET/CT data was spatially transformed into the matrix of th specific MRI using ridged matching. This protocol guarante the PET/CT data and exact anatomical mapping and was alr the generated atlas was overlaid with the PET data, and the vo OB) in the PET dataset were defined. The reference region (c the target receptor, and an internal control region (striatum MRI-based rat brain Schiffer atlas [158] (Figure 9D-F). T representing the changes in radioactivity concentration over TACs were used for kinetic modeling with the PKIN tool. Th the Simplified Reference Tissue Model 2 (SRTM2).

Atlas Generation and PET/CT Data Analysis
The D 2 /D 3 R availability was quantified with the software PMOD v3.7 (PMOD Technologies LLC, Zurich, Switzerland). A reference model was used, and the non-displaceable Binding Potential (BP nd ) was determined. For this purpose, the target regions (left and right OB) were delineated in a first step using a self-designed group-specific MRI atlas. For the generation of the atlas, the OB was defined in a representative MRI of the different experimental groups in each slice of the MRI dataset ( Figure 9A-C). A 3D-voxel (Schiffer space [158]) fitting the anatomical dimensions of the OB was calculated. The PET/CT data was spatially transformed into the matrix of the Schiffer space via the rat-specific MRI using ridged matching. This protocol guarantees high spatial resolution of the PET/CT data and exact anatomical mapping and was already published [58]. Finally, the generated atlas was overlaid with the PET data, and the voxels of interests (VOIS; here: OB) in the PET dataset were defined. The reference region (cerebellum), being devoid of the target receptor, and an internal control region (striatum) were delineated with the MRI-based rat brain Schiffer atlas [158] (Figure 9D-F). Time-activity curves (TACs) representing the changes in radioactivity concentration over time were extracted. These TACs were used for kinetic modeling with the PKIN tool. The BP nd was calculated using the Simplified Reference Tissue Model 2 (SRTM2).

Tract-Tracing-Based Generation of Connectome Data
To verify whether neuronal connections between the CPu and the main olfactory bulb (MOB) in the 6-OHDA lesion model and BoNT-A therapy approach could possibly be responsible for the improvement in smelling capability, a connectome analysis was performed. The basis for the rat nervous system connectome data is a complete meta-study of all tract-tracing original research publications imported into the neuroVIISAS framework [159]. The directional and weighted data were validated [160]. For connectomics, the MOB was analyzed to differentiate clearly from the connections of the accessory olfactory

Tract-Tracing-Based Generation of Connectome Data
To verify whether neuronal connections between the CPu and the main olfactory bulb (MOB) in the 6-OHDA lesion model and BoNT-A therapy approach could possibly be responsible for the improvement in smelling capability, a connectome analysis was performed. The basis for the rat nervous system connectome data is a complete metastudy of all tract-tracing original research publications imported into the neuroVIISAS framework [159]. The directional and weighted data were validated [160]. For connectomics, the MOB was analyzed to differentiate clearly from the connections of the accessory olfactory bulb. Since the term OB is usually used in the literature, instead of MOB, we also use OB here.

Statistical Analysis
In general, an overall significance level p = 0.05 was used. Normally distributed data were subjected to one-way ANOVA or two-way ANOVA using SigmaPlot 14 Software (Systat Software, Inc., San Jose, CA 95110, USA). In the case of statistically significant different mean values, data were subjected to all pairwise multiple comparison procedures (Holm-Sidak method).
If the normality test (Shapiro-Wilk) or equal variance test (Brown-Forsythe) failed, a Kruskal-Wallis one-way ANOVA was done on ranks. In the case of statistically significant different median values among the treatment groups, a multiple comparison procedure (Dunn's Method) was used.
To determine the strength of association of each behavioral test to apomorphine-or amphetamine-induced rotations, we performed correlation analyses. Prior to correlation,

Statistical Analysis
In general, an overall significance level p = 0.05 was used. Normally distributed data were subjected to one-way ANOVA or two-way ANOVA using SigmaPlot 14 Software (Systat Software, Inc., San Jose, CA 95110, USA). In the case of statistically significant different mean values, data were subjected to all pairwise multiple comparison procedures (Holm-Sidak method).
If the normality test (Shapiro-Wilk) or equal variance test (Brown-Forsythe) failed, a Kruskal-Wallis one-way ANOVA was done on ranks. In the case of statistically significant different median values among the treatment groups, a multiple comparison procedure (Dunn's Method) was used.
To determine the strength of association of each behavioral test to apomorphine-or amphetamine-induced rotations, we performed correlation analyses. Prior to correlation, the data were subjected to a Shapiro-Wilk normality test. In the case of normally distributed data, the Pearson product moment correlation test was done. The Pearson product moment correlation coefficient is a parametric test that does not require the variables to be assigned as independent and dependent. Instead, only the strength of association is measured. If the Shapiro-Wilk normality test failed (no normal distribution), Spearman rank-order correlation was performed. Spearman rank-order correlation is a nonparametric test that does not require the data points to be linearly related with a normal distribution about the regression line with constant variance. The Spearman rank-order correlation coefficient does not require the variables to be assigned as independent and dependent. Instead, only the strength of association is measured. Regression lines and prediction intervals were inserted into the resulting scatter plots. Prediction intervals, also called the confidence interval for the population, describe the range where the data values will fall a percentage of the time for repeated measurements.

Conclusions
We present here a longitudinal study of changes in D 2 /D 3 receptor availability in the olfactory bulb of the 6-OHDA-induced hemi-PD rat model and results for buried pellets tests. Because olfactory performance appears to be unchanged in hemi-PD rats, our results suggest that the 6-OHDA model may not be appropriate to study olfactory capacity in the context of dopaminergic neurodegeneration in the SNpc. Nevertheless, this study provides first insights into the changes in the olfactory system after intrastriatal BoNT-A injection in hemi-PD rats. In these 6-OHDA + BoNT rats, a relative interhemispheric increase in the availability of D 2 /D 3 receptors in the OB may be induced via indirect connectivity between the CPu and the OB.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/toxins14020094/s1, Figure S1: The latencies to find the pellet in the buried pellet test are correlated with the optical densities of the glomerular layers of the left OB of rats of the 6-OHDA + Sham and Sham + Sham groups (A), and the 6-OHDA + BoNT and 6-OHDA + Sham groups (C), and with the respective optical densities of the glomerular layers of the right OB of rats of the 6-OHDA + Sham and Sham + Sham groups (B), and the 6-OHDA + BoNT and 6-OHDA + Sham groups (D). Neither parameters showed significant correlations. Regression lines are displayed as solid lines and prediction intervals as dashed lines.; Figure S2: The optical densities of glomerular layers of the left OB are correlated with apomorphine-induced rotations of rats of the 6-OHDA + Sham and Sham + Sham groups (A), and 6-OHDA + BoNT and 6-OHDA + Sham groups (B). Optical densities are correlated with the respective amphetamine-induced rotations of rats of the 6-OHDA + Sham and Sham + Sham groups (C), and the 6-OHDA + BoNT and 6-OHDA + Sham groups (D). No significant correlations between these parameters were found. Regression lines are displayed as solid lines and prediction intervals as dashed lines.; Figure S3: The optical densities of the glomerular layers of the right OB are correlated with apomorphine-induced rotations of rats of the 6-OHDA + Sham and Sham + Sham groups (A), and the 6-OHDA + BoNT and 6-OHDA + Sham groups (B), and with the respective amphetamine-induced rotations of rats of the 6-OHDA + Sham and Sham + Sham groups (C), and the 6-OHDA + BoNT and 6-OHDA + Sham groups (D). No significant correlations between these parameters were found. Regression lines are displayed as solid lines and prediction intervals as dashed lines.  Center) for their tender care of the animals. The artwork of Sandra Maria Pietras, Macroscopic and Clinical Anatomy, Medical University of Graz, is gratefully acknowledged.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.