Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain

doi:10.21203/rs.3.rs-1967061/v1

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Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain

https://doi.org/10.21203/rs.3.rs-1967061/v1

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published 17 Jan, 2023

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Background: A historically definitive marker for cholinergic neurons is choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, can be found in high concentration in cholinergic neurons, both in the central and peripheral nervous systems. ChAT, produced in the body of the neuron and is transported to the nerve terminal, where its concentration is highest, catalyzes the transfer of an acetyl group from the coenzyme acetyl-CoA to choline, yielding acetylcholine (Ach). The creation of the bacterial artificial chromosome (BAC) transgenic mice expressing promoter-specific fluorescent reporter proteins (green fluorescent protein - GFP) provided an enormous advantage for neuroscience. Both in vivo and in vitro experimental methods benefited from the transgenic visualization of the cholinergic neurons. Mice were created by adding a BAC clone into the ChAT locus, in which eGFP is inserted into exon 3 at the ChAT initiation codon, robustly and supposedly selectively expressing enhanced GFP (eGFP) in all cholinergic neurons and fibers in the central and peripheral nervous systems, as well as in non-neuronal cells.

Methods: This project systematically compared the exact distribution of the ChAT-eGFP expressing neurons in the brain with the expression of ChAT by immunohistochemistry using mapping, and comparison with in situ hybridization (ISH).

Results: We qualitatively described the distribution of ChAT-eGFP neurons in the mouse brain comparing it with the distribution of immunoreactive neurons and ISH data, paying special attention to areas where the expression is not overlapping, such as the cortex, the striatum, the thalamus and the hypothalamus. We found complete overlap between the transgenic expression of eGFP and the immunohistochemical staining in the areas of the cholinergic basal forebrain. However, in the cortex and hippocampus we found small neurons that were only labelled with the antibody and not expressed eGFP or vice versa. Most importantly, we found no transgenic expression of eGFP in the lateral dorsal, ventral and dorsomedial tegmental nuclei cholinergic cells.

Conclusion: While the majority of the forebrain choline acetyltransferase expression was aligned in the transgenic animals with immunohistochemistry, other areas of interest, such as the brainstem should be considered before choosing this particular transgenic mouse line.

transgenic mice

ChAT eGFP

immunohistochemistry

basal forebrain cholinergic neurons

antibody staining

in situ hybridization

Allen Mouse Brain Atlas

choline acetyltransferase

Since the generations of the first transgenic mouse line in 1989 by Gordon et al., the technique of introducing a foreign gene into a mouse oocyte using pronuclear injection has become widely spread, resulting in the generation of hundreds of different mouse lines (Gordon et al., 1989). Thus, with meticulous technique, any cloned gene sequence could be integrated into the DNA of rodents, and once injected these genes insert into the host DNA at random sites, being dispersed throughout the genome. Due to the development in molecular biological techniques and genetics, genes can be inserted, deleted, multiplied, and conditionally expressed. Combined with another revolutionary development in neuroscience, the expression of green fluorescent protein (GFP) in the mammalian brain (Chiocchetti et al., 1997), the scientific community got ready access to visualize certain cell types in the vertebrae nervous system without using immunohistochemistry. GFP is a naturally fluorescent gene product of the bioluminescent jellyfish Aequorea victoria. Since the native GFP protein is not particularly bright (excited by UV light at 395 nm) scientists were more interested in the genetically engineered versions, such as the enhanced GFP (eGFP), with red-shifted excitation maxima (induced using lasers emitting at 488 nm) and increased quantum efficiencies (up to 35-fold) (Kusser and Randall, 2003). Thus, the eGFP version was much more useful in fluorescence and confocal microscopy. These techniques without a doubt contributed to significant discoveries about the structure and function of the central nervous system. Today there are 644 mouse strains available in stock at Jacksons Laboratories containing a fluorescent protein insert (The Jackson Laboratory, 2022).

The basal forebrain (BF) cholinergic system has been in the center of attention in the past 30 years, first because of its role in the regulation of sleep-wake cycle and cortical activation, and because of its possible connection with Alzheimer’s disease and other dementias (Whitehouse et al., 1982; Szymusiak and McGinty, 1986; Vana et al., 2011; Zant et al., 2012; Szutowicz et al., 2013). Cholinergic neurons were traditionally visualized by immunohistochemistry, by using antibodies against choline-acetyltransferase (ChAT), which is essential for acetylcholine (ACh) synthesis (Ishida et al., 1983; Levey et al., 1983). However, the recent bloom in the creation of transgenic animals in the past decades emerged as a powerful new tool in visualizing certain type of neurons or proteins. The cloning of the ChAT^BAC- eGFP mouse not only allowed scientists to immediately visualize the cholinergic cell population under epifluorescent light, but also made targeted electrophysiological recordings and embryological, developmental examinations possible (Fukushima et al., 2011; Kramer et al., 2011; Muranishi and Furukawa, 2012). The ChAT^BAC- eGFP mouse is widely used now the investigation of central nervous system, spinal cord motor and interneurons in adults and embryos, retinal cells and auditory tube (Miller et al., 2009; Mesnage et al., 2011; Krasteva et al., 2012; Wu et al., 2012). The bacterial artificial chromosome (BAC) transgenic approach utilizing the ChAT locus to direct expression of eGFP has been described (Ju et al., 2003; Tallini et al., 2006). However, the marker expression in peripheral cholinergic neurons was not reported in these mice, preventing determination of the effectiveness of the BAC strategy in the periphery.

Up to now, a comprehensive report about the distribution of ChAT promoter driven eGFP expression in the entire mouse brain has not been described in detail. To validate the expression of ChAT-eGFP in the entire mouse brain, here we report the precise qualitative distribution of neurons expressing the inserted eGFP and compare that distribution to the traditional immunohistochemical method of labeling ChAT, as well as with the in situ hybridization of the ChAT mRNA expression published by the Allen Mouse Brain Atlas (Allen reference atlas – mouse brain [gene search], 2004).

To investigate the distribution of ChAT-eGFP positive cells in the adult mouse brain, we individually mapped and analyzed series of sections from the entire mouse brain for neurons that contain ChAT visualized either by transgenic eGFP protein expression, immunohistochemistry or in situ hybridization (ISH), published by the Allen Institute for Brain Science (Allen reference atlas – mouse brain [gene search], 2004). We manually mapped 44 sections starting from + 2.40 mm from the bregma to -6.4 mm using MBF Neurolucida software, covering the entire brain in coronal sections and compared the localization and expression pattern of the cholinergic neurons expressing the eGFP tag, antibody or choline acetyltransferase RNA.

Forebrain regions that contain ChAT positive neurons

Telencephalon

Scattered pattern of both ChAT-eGFP and Gt-a-ChAT labeled neurons were found throughout the prefrontal cortical areas. ChAT-eGFP neurons were concentrated in the anterior olfactory area (AO) medial and posterior parts as well as in the endopiriform claustrum (En). Both ChAT-eGFP and antibody labeled neurons were sparsely dispersed in the cortical areas, including the orbital (O), the prelimbic (PrL), infralimbic (Il), cingulate (Cg), motor (M), somatosensory (S), insular (I) and dense cell layer piriform (Pir) cortices. In the cortical areas, the two markers showed very low overlap at any levels (Fig. 1A; Fig. 3A-C).The dorsal tenia tecta (DTT) contained ChAT-eGFP neurons, but no antibody labelled neurons (Fig. 1B). Consistent with the observations, ISH data represented similar scattered ChAT mRNA expression in the isocortex and olfactory areas (OLF), as shown in comparative right-hand side image panels in Fig. 1.

The medial septum (MS), the nucleus of the horizontal, ventral and lateral limb of the diagonal band (HDB/VDB/LDB), ventral pallidum (VP), the caudate putamen (CPu), the globus pallidus (GP), and the extension of the amygdala (EA) only contained double labeled neurons in their entire expansion (Fig. 1C-D; Fig. 3D-I). We also found double-labelled neurons in the tuberculum (Tu) (Fig. 1C-D). Throughout the accumbens nucleus (ACB) shell ChAT -eGFP/Gt-a-ChAT positive double-labelled neurons were found (Fig. 1C-D). Consistent with the observations, ISH data represented similar high ChAT mRNA expression in the entire basal forebrain expansion, Tu, ACB shell, and striatum as shown in comparative right-hand side image panels in Fig. 1.

Occasionally, ChAT-eGFP neurons were found in the hippocampus cornu ammonis (CA1-3), dentate gyrus (DG) regions, but the appearance of the positively antibody labelled cells were extremely sporadic (Fig. 1E-F; Fig. 2A-B; Fig. 4A-I). ISH ChAT mRNA expression levels were consistent with ChAT-eGFP expression patterns in the hippocampus, as shown in comparative right-hand side image panels in Fig. 1E-F and Fig. 2A-B.

Among the amygdaloid regions, the medial amygdala (Me) including the anterior (MeA), posterodorsal (MePD), posteroventral (MePV) and the basomedial amygdala nucleus (BMA) were labelled with ChAT-eGFP but not with the antibody (Fig. 1E-F, Fig. 2A). ISH mRNA expression was high in the amygdaloid regions and was similar to ChAT-eGFP expression patterns as shown in comparative right-hand side image panels in Figs. 1 and 2.

Diencephalon

Significant number of ChAT-eGFP positive neurons were located at the LOT2, and they were not detected by using the antibody. Besides the basal forebrain areas, the medial habenula (MHb) also contained double-labelled neurons, but interestingly the lateral habenula (LHb) only contained neurons labelled with ChAT-eGFP in the transgenic mice (Fig. 1F). Consistent with ChAT-eGFP expression, ISH data shows ChAT mRNA expression in both MHb and LHb.

In the thalamus, ChAT-eGFP neurons can be found in the parafascicular (Pf), posterior (Po), pretectal (PoT) and posteromedial areas (PoM) (Fig. 2A). In the zone incerta (ZI), which is an extension of the reticular nucleus of the thalamus, we found high number of ChAT-eGFP positive neurons throughout the structure, but no overlap with the antibody reaction (Fig. 1E). ISH data was consistent with this observation indicating ChAT mRNA expression in the thalamic expansion including the ZI (Fig. 1E-F; Fig. 2A-B).

The preoptic areas and the hypothalamus were heavily populated with small, ChAT-eGFP neurons that were not detected by the antibody. In the hypothalamus, the lateral anterior hypothalamus (LAH), ventrolateral hypothalamus (VLH), anterior hypothalamic nucleus (AHC), lateral hypothalamus (LH) and posterior lateral hypothalamus (PLH) areas are strongly ChAT-eGFP labeled (Fig. 1E-F; Fig. 2A-B; Fig. 5G-I). We also have observed ChAT mRNA expression in these hypothalamic regions. In the arcuate nucleus (Arc) we found neurons labeled by only the antibody but not the eGFP (Fig. 1F). ISH data was consistent with the antibody labelling and we observed ChAT mRNA expression in the Arc.

Midbrain and Hindbrain regions that contain ChAT positive neurons

Double labelled and ISH ChAT mRNA expressing neurons were located in the pedunculotegmental areas (PTg), in the parabigeminal nucleus (PBG) (Fig. 2C; Fig. 5D-F), the cranial nerve nuclei, including the oculomotor nucleus (3N), the motor trigeminal nucleus (5N) (Fig. 2D-E; Fig. 6D-F), the facial nucleus (7N), the abducens nucleus (6N), the dorsal motor nucleus of the vagus (10N), and the hypoglossal nucleus (12N) (Fig. 2D-E). The nucleus ambiguous (NA) also contained both eGFP and immunoreactive ChAT antibody labelled cells (Fig. 2E-F).

A population of ChAT-eGFP and ISH ChAT mRNA expressing neurons was found in the inferior colliculus (CIC), but not labelled by the antibody (Fig. 2D; Fig. 5A-C).

We found areas that revealed cholinergic neurons by using immunohistochemistry and ISH only in the lateral dorsal (LDT) and ventral (VT) tegmental nucleus (Fig. 2D). A small population of ChAT-eGFP expressing neurons was found in the dorsomedial tegmental nucleus (DMT) (Fig. 2D; Fig. 6A-C).

We also found both ChAT-immunostained and ChAT-eGFP expressing neuronal population in the mesencephalic trigeminal nucleus (Me5) consistent with ISH ChAT mRNA expression data (Fig. 2D; Fig. 6G-I).

Summary of anatomical areas with ChAT-eGFP and antibody expression

In summary, almost perfect overlap in the basal forebrain and striatal areas, more precisely in the MS, VDB/HDB/LDB, VP, EA, and in the CPu, GP, and the interstitial nucleus of the posterior limb of the anterior commissure (IPAC) (Fig. 1C-D; Fig. 3D-F). Few regions such as the PLH, PBG, 5N, and Me5 had cells with antibody labelling or eGFP expression only as well as overlap of both. Moreover, both antibody labelling and ChAT-eGFP expression in transgenic mice were consistent with ISH ChAT mRNA expression data.

To summarize our results, we found prominent ChAT-eGFP expression, without corresponding ChAT-antibody binding, in the preoptic areas, including DTT, lateral septum, and LHb. We found occasional but consistent number of neurons in the hippocampus (CA1-CA3, DG) (Fig. 4A-I), thalamus, periaqueductal grey, and vestibular nucleus. The CIC of the midbrain (Fig. 2D; Fig. 5A-C) and the DMT of the midbrain nuclei (Fig. 2D; Fig. 6A-C) also showed significant ChAT-eGFP expressing neurons.We observed high intensity antibody labelling of ChAT neurons in the LDT, as well as a secluded sub population of cells in Me5 only expressing ChAT immunoreactive cells (Fig. 6G-I).

Table 1

Qualitative summary of brain regions expressing ISH ChAT mRNA, along with either ChAT-eGFP or ChAT immunostaining.
Region of the brain	Corresponding figures	ChAT-eGFP	Gt-a-ChAT
Forebrain
Olfactory bulb (Olf)
Anterior olfactory area (AO)	Figure 1A-B	+	+
Medial olfactory area (MO)	Figure 1A-B	+	+
Posterior olfactory area (PO)	Figure 1A-B	+	+
Endopiriform claustrum (En)	Figure 1A-B	+	+
Neocortex/ Isocortex
Orbital (O)	Figure 1A-B	++	+
Prelimbic (PrL),	Figure 1A-B; Fig. 3A-C	++	+
Infralimbic (Il),	Figure 1A-B	++	+
Cingulate (Cg),	Figure 1A-B	++	+
Motor (M),	Figure 1A-B	++	+
Somatosensory (S),	Figure 1A-B	++	+
Insular (I)	Figure 1A-B	++	+
Dense cell layer piriform (Pir)	Figure 1A-B	++	+
Dorsal tenia tecta (DTT)	Figure 1A-B	++	-
Basal ganglia and septum
Medial septum (MS)	Figure 1C; Fig. 3D-F	++++	++++
Horizontal limb of the diagonal band (HDB)	Figure 1C-D	++++	++++
Ventral limb of the diagonal band (VDB)	Figure 1C-D	++++	++++
Lateral limb of the diagonal band (LDB)	Figure 1C-D	++++	++++
Ventral pallidum (VP)	Figure 1C-D	++++	++++
Caudate putamen (CPu)	Figure 1C-D	+++	+++
Globus pallidus (GP)	Figure 1C-D	+++	+++
Extension of the amygdala (EA)	Figure 1C-D	+++	+++
Tuberculum (Tu)	Figure 1C-D	+++	+++
Accumbens nucleus shell (ACB)	Figure 1C-D	+++	+++
Hippocampus
Cornu ammonis (CA)	Figure 1E-F; Fig. 2A; Fig. 4A-F	++	-
Dentate gyrus (DG)	Figure 1E-F; Fig. 2A; Fig. 4G-I	++	-
Amygdaloid regions
Medial amygdala (Me)	Figure 1E-F; Fig. 2A	++	-
Anterior medial amygdala (MeA)	Figure 1E-F; Fig. 2A	++	-
Posterodorsal medial amygdala (MePD)	Figure 1E-F; Fig. 2A	++	-
Posteroventral medial amygdala (MePV)	Figure 1E-F; Fig. 2A	++	-
Basomedial amygdala nucleus (BMA)	Figure 1E-F; Fig. 2A	++	-
Diencephalon
Medial habenula (MHb)	Figure 1F	+++	+++
Lateral habenula (LHb)	Figure 1F	+++	-
Parafascicular thalamic nucleus (Pf)	Figure 1E-F; Fig. 2A-B	++	++
Posterior thalamic nucleus (Po)	Figure 1E-F; Fig. 2A-B	++	++
Pretectal thalamic nucleus (PoT)	Figure 1E-F; Fig. 2A-B	++	++
Posteromedial thalamic nucleus (PoM)	Figure 1E-F; Fig. 2A-B	++	++
Zone incerta (ZI))	Figure 1E-F; Fig. 2A-B	++	++
Lateral anterior hypothalamus (LAH)	Figure 1E-F; Fig. 2A-B	++	++
Ventrolateral hypothalamus (VLH)	Figure 1E-F; Fig. 2A-B	++	++
Anterior hypothalamic nucleus (AHC)	Figure 1E-F; Fig. 2A-B	++	++
Lateral hypothalamus (LH)	Figure 1E-F; Fig. 2A-B	++	-
Posterior lateral hypothalamus (PLH)	Figure 1E-F; Fig. 2A-B; Fig. 5G-I	++	++
Arcuate nucleus (Arc)	Figure 1E-F; Fig. 2A-B	-	++
Midbrain and Hindbrain
Pedunculotegmental areas (PTg)	Figure 2C	+++
Parabigeminal nucleus (PBG)	Figure 2C; Fig. 5D-F	+++	+
Oculomotor nucleus (3N)	Figure 2D-E	+++	++
The motor trigeminal nucleus (5N)	Figure 2D-E; Fig. 6D-F	+++	++
The facial nucleus (7N)	Figure 2D-E	+++	++
The abducens nucleus (6N)	Figure 2D-E	+++	++
The dorsal motor nucleus of the vagus (10N)	Figure 2D-E	+++	++
The hypoglossal nucleus (12N)	Figure 2D-E	+++	++
Inferior colliculus (CIC)	Figure 2D; Fig. 5A-C	+++	-
Dorsomedial tegmental nucleus (DMT)	Figure 2D; Fig. 6A-C	++	-
Laterodorsal tegmental nucleus (LDT)	Figure 2D; Fig. 6A-C	+	++
Ventral tegmental nucleus (VTg)	Figure 2D	+	++
Mesencephalic trigeminal nucleus (Me5)	Figure 2D; Fig. 6G-I	+	++

NOTE

Only areas demonstrating ISH ChAT mRNA expression are listed

Expression of ChAT-eGFP expression and Gt-a-ChAT labelling was estimated qualitatively based on both number of labeled cells and signal strength: ++++, highest density; +++, high density; ++, moderate density; +, low density; -, background density

Immunohistochemistry is an important and widely used application to identify cholinergic neurons. The enzyme choline acetyltransferase (ChAT) acetylates choline to acetylcholine (Ach) and has been successfully used as a marker to specifically stain cholinergic neurons in several immunohistochemical studies (Mesulam et al., 1983b, 1983a). However, this approach seems to have some limitations, and to address these inconsistencies the transgenic mice that express eGFP directed by the promoter for ChAT was developed. The ChAT^BAC- eGFP experimental mouse model facilitates the identification and study of the cholinergic nerves in the central and peripheral nervous systems (Tallini et al., 2006). However, we identified discrepancies between Chat-eGFP expression and immunostaining in several different important brain regions such as the forebrain, diencephalon, and the midbrain when compared to the immunohistochemical presentation of the ChAT antibody. We manually mapped and qualitatively analyzed coronal sections of the entire mouse brain for the expression of neurons that contain ChAT, mapping both ChAT-eGFP and ChAT antibody labelled cells. These expression patterns were then compared with Allan Brain Institute images of ChAT in situ hybridization on adult mouse brain. We have also included a qualitative summary of all our findings in Table 1.

The transgenic mice, ChAT^BAC- eGFP, used in this study was created using the strategy mentioned by Tallini et al. (2006). This new strategy employed a bacterial artificial chromosome (BAC) construct spanning the entire mouse cholinergic gene locus and an approximately 78 kb of 3’- and 36 kb of 5’-flanking DNA. Within this construct eGFP was knocked into the 3rd exon to replace the start codon of the ChAT gene and was linked to a poly-A signal that terminates translation of the mRNA into protein. Hence, in these mice the transcription of transgenic ChAT is impeded and replaced by eGFP. It is noteworthy that ChAT-eGFP mice have four genomic copies of the cholinergic gene locus, which contains the vesicular acetylcholine transporter (VAChT) and ChAT promoter and coding regions (Tallini et al., 2006; Nagy and Aubert, 2012). However, it was confirmed that a single copy of the ChAT gene was expressed like in the wild-type mice, as the ChAT protein levels were not statistically different in the brain of ChAT-eGFP mice compared to wild-type mice, while the GFP mRNA and protein expression is two-three times higher than ChAT expression (Nagy and Aubert, 2012, 2013). Moreover, while the transcription of the VAChT transgene remains operational; VAChT is overexpressed, while levels of ChAT, choline transporter (CHT), and acetylcholinesterase (AChE) are maintained, in cholinergic neurons (Nagy and Aubert, 2012, 2013). Accordingly, the ChAT-eGFP mouse model validate the use of GFP fluorescence expression as an indicator for the presence of ChAT.

It is not yet confirmed whether the lack of complete expression fidelity in this line, especially in the peripheral nervous system, is related to lack of distinct transcriptional control/ regulatory process due to random insertion of a very large piece of DNA into the mouse genome. That is, BAC transgene may not necessarily be inserted in or near the gene locus of interest thus the exact insertion site is still unknown. There is also the possibility of the entire BAC spanning the ChAT locus might have not been inserted due to rearrangements or truncations. Such alterations could alter or remove some cis regulatory modules, including cell type specific enhancer binding sites, possibly explaining the lack of eGFP expression in some cholinergic cell classes that might be exploiting a somewhat distinct transcriptional regulatory process. A second possibility is that, since fluorescence requires active transcription at the time of analysis as well as sufficient transcription to be able to detect the eGFP, these cells are less transcriptionally active and/or that the Ach receptor protein is stabilized, requiring less robust protein production. We also identified brain regions with little or no antibody immunoreactivity but with marked eGFP expression. ChAT is synthesized in the neuronal soma and transported to neuronal varicosities where synthesis, storage, and release of ACh occurs. Thus, the relative abundance of cholinergic marker level in neuronal soma would depend on the rate of synthesis, type of neuronal sub population, and brain region, affecting the distribution of goat-anti-ChAT immunoreactive neurons in a given region.

In this study, we reported striking discrepancies between ChAT-eGFP and Goat-anti-ChAT labelling in the mouse cortical brain areas. According to Figs. 3A-C, there is a scattered distribution of ChAT⁺ neurons throughout the entire cortex, with ChAT-eGFP positive neurons outnumbering antibody labelling and scarce overlap of the two markers. Consistent with previous findings showing that in the cortex colocalization and labelling is lower than in other brain regions (Von Engelhardt et al., 2007). Presence of putative cholinergic neurons in the rodent cortex was first identified through immunolabelling for ChAT (Ecicenstein and Thoenen, 1983). Even theses early studies emphasized the limitations in staining sensitivity of these ChAT-immunoreactivity containing neurons throughout the entire cortex, compared to cholinergic neurons in the basal forebrain. Thus, confirming the reliability of transgenic rodent models with intrinsic eGFP expression specific to ChAT, in giving reproducible access to analyze the physiology and function of cortical cholinergic neurons. Especially for electrophysiological/ cell isolation/ tissue culture studies of cortical cells and circuits, and for crucial imaging (Gong et al., 2003). Furthermore, it has been reported that anti-ChAT antibody is quite sensitive to the fixative PFA, ensuing very weak immunoreactivity in cells with scarce ChAT content such as cortical interneurons (Von Engelhardt et al., 2007). Additionally, as seen in Fig. 3A-C, we observed a strong immunoreactivity of a dense network of fibers in the cortex, most likely formed of axons originating from basal forebrain cholinergic neuronal population, which could be masking the weakly ChAT-immunoreactive cell bodies. When visualized, we observed that cortical eGFP-positive neurons cluster mainly into two groups, bipolar and multipolar cells as previously reported (Von Engelhardt et al., 2007) with bipolar appearance constituting most eGFP-positive interneurons (Fig. 3A-C). Furthermore, recent studies have emphasized the importance of genetically labelled cholinergic neurons in rodents to study different subpopulations of the cortical interneurons, to provide quantitative information on neuronal distribution and detail patterns of axonal projections (Li et al., 2017; Záborszky et al., 2018; Granger et al., 2020).

The most striking colocalization of Chat-eGFP expression and anti-Chat immunoreactivity was seen in the basal forebrain (BF) cholinergic neurons. These results were consistent when compared to Allan Brain Institute in situ hybridization maps (Fig. 1C-D). Moreover, the BF is composed of various structures including the extended amygdala (EA) and peripallidal regions, facilitating processes of cortical activation motivation, attention, learning, and memory (Mesulam et al., 1983a; Masuda et al., 1997; Kanemoto et al., 2020). The BF is largely populated by cholinergic neurones compared to other neuronal cell types in rodents and primates, and these neurones project to the cerebral cortex, the hippocampus, and the amygdala (Gritti et al., 2003; Záborszky et al., 2018). We found virtually all eGFP-positive neurons are also ChAT-immunoreactive in the entire BF expansion including the MS, VDB/HDB/LDB, VP, EA (Fig. 3D-I). Hence, confirming that the transgene recapitulated faithfully the expansion of the endogenous gene, predominantly in areas where ChAT levels are high, also including the CPu, GP, IPAC (Fig. 1). Our findings were also consistent with previous reports using ChAT-eGFP (Von Engelhardt et al., 2007) and ChAT-tauGFP/ ChAT-YFP (Yi et al., 2015) transgenic mice.

According to our observations, eGFP expression could be observed in all areas known to contain cholinergic neurons. In our initial examination of the hippocampus of ChAT-eGFP mice, dense network of GFP-containing fibers were found innervating all hippocampal layers, presumably arising mainly from MS-DBB cholinergic projection neurons of the basal forebrain (Dutar et al., 1995). In contrary, this network was not as dense and significant with antibody labelling (Fig. 4). Moreover, intrinsic hippocampal cholinergic interneurons have been found almost 30 years ago, that may contribute to an intrinsic pool of ACh (Frotscher et al., 1986, 2000). Consistent with these findings we observed a sparse and scattered distribution of eGFP-expressing cholinergic interneurons in the CA1, CA3, and DG subregions, though not as big and bright as the basal forebrain neurons (Fig. 4). Interestingly, we could not observe these neurons when stained with goat-anti-ChAT antibody, most likely due to low signal-to-noise ratio of ChAT antibody labelling showing very weak ChAT immunoreactivity in the hippocampus compared to that in the basal forebrain (Frotscher et al., 1986). More recent findings also confirm these observations, reiterating that eGFP (Gong et al., 2007; Von Engelhardt et al., 2007; Grybko et al., 2011) or eYFP (Yi et al., 2015) expression under the control of the ChAT promotor is likely to amplify the detection sensitivity of ChAT expressing cells beyond the detection sensitivity of anti-ChAT antibody. Further confirming ChAT-eGFP transgenic mouse model is a reliable model to investigate the physiological and functional properties of hippocampal cholinergic interneurons.

The inferior colliculus (CIC), a midbrain hub for both ascending and descending auditory pathways, contains nicotinic and muscarinic cholinergic receptors throughout, and ACh modulates the responses to acoustic stimuli of a majority of CIC cells (Beebe and Schofield, 2021). The CIC receives cholinergic inputs mainly from the pedunculopontine tegmental nucleus (PPT) (Noftz et al., 2020). However, reports of intrinsic cholinergic neuronal cell population within the CIC are almost none. We report a significant population of ChAT-eGFP expressing neurons in the CIC (Fig. 5A-C) but not labelled by the antibody.

It was first discovered in 1986 the presence of smaller cholinergic populations (Ch8) in the parabigeminal nucleus (PBG) located at the lateral edge of the midbrain (Mufson et al., 1986). It was also reported that in the mouse approximately 80–90% of the PBG cells are cholinergic providing a major source of extrinsic cholinergic projections to the superior colliculus (Mufson et al., 1986; Tokuoka et al., 2020). Similarly, in our study, we observed both Chat-eGFP expression as well as antibody labelling in the PBG. However, as we see an overlap of both eGFP and antibody labelling, there were also some cells labelled only with the antibody towards the ventrolateral end of the cholinergic population (Fig. 5D-F). PBG contains neurons that lack VCHAT (Sokhadze et al., 2022) and this might be the reason for some ChAT neurons not expressing eGFP.

Smaller cholinergic populations are also located in the hypothalamus (Rao et al., 1987; Ahmed et al., 2019). A substantial proportion of hypothalamic cells were found in the lateral preoptic-hypothalamic continuum - peduncular part of the lateral hypothalamus (LPO-PLH) (Gielow and Zaborszky, 2017). In agreement with previous findings, we also report small population of sparingly distributed cholinergic neurons in the PLH (Fig. 5G-I). Here we found both ChAT-eGFP expressing and ChAT-immunoreactive cells, with some cells overlapping and some cells expressing only eGFP or Gt-anti-ChAT-labelling. Again, the differences most likely accounting for regional differences and expression fidelity.

The habenula is a complex nucleus, which is a part of the epithalamus connecting the limbic forebrain and the midbrain, and is divided into medial (MHb) and lateral (LHb) subregions (Gould et al., 2019). The MHb has drawn attention recently for its dense population of cholinergic neurons and the expression of unique nicotinic acetylcholine receptors (β4 and α5 subunits), thus thought to regulates nicotine aversion and withdrawal (López et al., 2019; Cho et al., 2020). In the current study, we observed doubled labelled neurons with eGFP expression and antibody labelling in the MHb, but lack of antibody labelling in the LHb (Fig. 1F). However, the LHb has received considerable attention for its potential role in cognition and pathogenesis of various psychiatric disorders, and since we observed eGFP expression in the LHb, the ChAT-eGFP mouse model could be a potential transgenic model to study further on the hebenula complex.

Cholinergic neurons of the midbrain nuclei, such as pedunculopontine nucleus (PPN) and laterodorsal tegmental nucleus (LDT), provide widespread innervation to the thalamus and the basal ganglia (Steriade et al., 1988; Huerta-Ocampo et al., 2020). Thus have been associated with locomotion, reward, arousal, and control of the sleep/wake cycle (Motts and Schofield, 2010; Xiao et al., 2016). In our study, in agreement with previous findings (Steriade et al., 1988; Spann and Grofova, 1992; Huerta-Ocampo et al., 2020, 2021), we report Chat-immunoreactivity in the LDT region of the midbrain with faint and sparse distribution of eGFP expression (Fig. 6A-C). However, for the first time, we found strong but small ChAT-eGFP expressing neurons in the dorsomedial tegmental (DMT) area (Fig. 6A) but interestingly no Goat-anti-ChAT immunoreactivity (Fig. 6B), accounting for regional specific expression fidelity of eGFP. Hence, Chat-eGFP mouse model could be useful in studying the cholinergic system in the DMT.

In our study, we observed the motor nucleus of the trigeminal-V (5N) densely packed with cholinergic neurons, as reported recently for Cre-dependent fluorescence reporter mouse line (Li et al., 2017). It is clear from the current study that eGFP expression and antibody labelling overlap for cholinergic neurons in the 5N for ChAT-eGFP mouse line, except for some sporadically distributed cholinergic cells with only eGFP expression as shown in Fig. 6D-F. The reason for these discrepancies could be fidelity of the ChAT-eGFP expression compared to goat-anti-ChAT immunoreactivity.

The mesencephalic trigeminal nucleus (Me5), located at the mesopontine junction, contains primary sensory neurons that innervate the muscle spindle of the masticatory muscles in the oro-facial region and is responsible for receiving and transmitting proprioception from this region (Wang et al., 2007). There are not many reports about the cholinergic neurons of the Me5. However, two reports on rats, state that Me5 contains prominent AChE-reactive cells (Paxinos et al., 2012) and ChAT-immunostained neurons (Spann and Grofova, 1992). In consistent with these studies, we report both ChAT-eGFP expression and Goat-anti-ChAT immunoreactivity in the Me5 region. There was a prominent aggregated neuronal population containing both eGFP and ChAT-immunoreactivity (Fig. 6G-I), but we also found a secluded population containing only ChAT-immunoreactivity (Fig. 6H-I). Once again accounting for regional specific eGFP expression.

In line with these findings, the ChAT^BAC- eGFP mouse model qualifies as a reliable transgenic model to study the cholinergic cell population, especially the cortical cholinergic interneurons, stratal cholinergic interneurons, and BF cholinergic projection neurons (Ch1-Ch4). Our study also provides evidence of a significant population of cholinergic interneurons in the hippocampal CA1, CA3 and DG regions visualised through eGFP expression. Thus, confirming the reliability of ChAT^BAC- eGFP transgenic mouse model to study the physiological and functional properties of hippocampal cholinergic interneurons. Our findings further suggest the reliability of this mouse model to study the mesopontine tegmentum (Ch5 and Ch6), medial habenula nucleus (Ch7), and parabigeminal nucleus (Ch8) cholinergic cell population. Even though, we found eGFP expression in areas such as the 5N and Me5, due to discrepancies in fidelity of eGFP expression compared to antibody labelling, using this model for areas such as the brainstem should be reconsidered. However, since the cholinergic system plays an essential role in several pathological conditions, there is no doubt that transgenic mouse models such as the one described in this study will help in the understanding the nature of cholinergic transmission in the brain and mechanistic information of influence of therapeutic interventions on this system.

Animals

The experiments were performed on adult ChAT^BAC- eGFP transgenic mice (JAX 00790, B6.Cg-Tg(RP23-268L19-EGFP)2Mik/J) JAX of mixed sex purchased from JAX (The Jackson Laboratory, Maine, USA), bred and housed at Western Sydney University, School of Medicine Animal Facility (Campbelltown, Australia). The creation of the transgenic animals was described elsewhere (Tallini et al., 2006). Mice were housed in a temperature-controlled environment, with a normal 12:12 h light-dark cycle, with free access to food and water. All mice were monitored daily. All procedures were in accordance with the regulations of the Animal Care and Ethics Committee of Western Sydney University (A14717).

Male adult mice (n = 4) were euthanized with sodium pentobarbitone (0.1 ml, 200 mg/ml) and transcardially perfused with ice-cold saline followed by 4% paraformaldehyde in 0.1M PBS (100 mL with 4mL/min rate). Brains were removed and stored in the same fixative at 4◦C for 6 hrs, and then transferred to 30% sucrose in 0.1M PBS overnight for cryoprotection. Once the brains sank to the bottom of the container, they were embedded and frozen in 4% gelatin in 0.1M PBS. Using a Leica CM 1950 cryostat, brains were cut into 40 µm thick coronal sections in three series. One series of sections of the whole brain were collected on gelatin-coated slides, the second and third series was collected in 0.1M PBS buffer for further immunohistochemical processing.

We used the second series of section for immunohistochemical labeling of ChAT. Sections were incubated with normal serum to prevent non-specific binding [3% normal donkey serum (Jackson Immunoresearch Laboratories, West Grove, PA) in PBS, 2 hours]. To visualize ChAT-positive neurons for fluorescent microscopy analysis, the sections were incubated with a goat-anti-ChAT primary antibody (1: 250, Millipore, #3429630, for 48 hours at 4 ͦC) with 0.1% Triton X-100 followed by AF 594 donkey-anti-goat IgG secondary antibody (1:200, Jackson Immunoresearch Laboratories, #129–709, for 2 hours at room temperature). The sections were then mounted on gelatin-coated slides and immediately coverslipped with VECTASHIELD antifade mounting medium with DAPI (Vector Laboratories, #H1200).

Mapping

One in three series of 40 µm sections through the adult brain was examined. Forty-four coronal sections were mapped by using a Zeiss AxioImager M2 microscope with Apotome (Carl Zeiss MicroImaging). Neurons expressing eGFP in cholinergic neurons of the transgenic mouse (green) and immunoreactive for ChAT (red) were mapped by Neurolucida (MBF Neurosciences, VT, USA). Neurons containing both eGFP and ChAT antigen were marked with blue.

In situ – Allan Brain Institute

We downloaded and analyzed images of ChAT in situ hybridization on adult mouse brain. Images were downloaded from the Allan Brain Institute website (http://www.brain-map.org/) and modified in contrast by using Adobe Lightroom 2022 (Adobe).

Confocal imaging

Cover slipped ChAT-eGFP samples stained for ChAT⁺ cholinergic cells were imaged using a Confocal ZEISS Laser Scanning Microscope (LSM800) with an argon laser and processed using the Zen Blue® software package. The super resolution images were obtained using Airyscan with 1.3× crop area, and Z-stacks (18 slices; 23.63 µm) were captured using a 20×/0.8 objective and NA1.0 for reflective imaging, (unless otherwise specified). Reflective imaging was achieved using the 561 nm wavelength for ChAT⁺ cells, and 488 nm wavelength for ChAT-eGFP expression. Extended depth of focus images were obtained by collapsing the Z-stacked 3D images of resolution 2146 × 2146 pixels, to better identify the objects and provide greater accuracy.

ABBREVIATIONS USED IN THE GENERAL TEXT

BAC	bacterial artificial chromosome
GFP	green fluorescent protein
eGFP	enhanced green fluorescent protein
ISH	in situ hybridization
DNA	deoxyribonucleic acid
ChAT	choline-acetyltransferase
ACh	acetylcholine
VAChT	vesicular acetylcholine transporter
PFA	paraformaldehyde

ABBREVIATIONS OF BRAIN REGIONS

AO	anterior olfactory area
En	endopiriform claustrum
O	orbital cortex
PrL	prelimbic cortex
Il	infralimbic cortex
Cg	cingulate cortex
M	motor cortex
S	somatosensory cortex
I	insular cortex
Pir	piriform cortex
DTT	dorsal tenia tecta
OLF	olfactory areas
BF	basal forebrain
MS	medial septum
HDB	horizontal limb of the diagonal band
VDB	ventral limb of the diagonal band
LDB	lateral limb of the diagonal band
(VP)	ventral pallidum
(CPu)	caudate putamen
(GP)	globus pallidus
(EA)	extension of the amygdala
(Tu)	tuberculum
(ACB)	accumbens nucleus shell
CA	cornu ammonis
DG	dentate gyrus
Me	medial amygdala
MeA	anterior medial amygdala
MePD	posterodorsal medial amygdala
MePV	posteroventral medial amygdala
BMA	basomedial amygdala nucleus
MHb	medial habenula
LHb	lateral habenula
Pf	Parafascicular thalamic nucleus
Po	posterior thalamic nucleus
PoT	pretectal thalamic nucleus
PoM	posteromedial thalamic nucleus
ZI	zone incerta
LAH	lateral anterior hypothalamus
VLH	ventrolateral hypothalamus
AHC	anterior hypothalamic nucleus
LH	lateral hypothalamus
PLH	posterior lateral hypothalamus
Arc	arcuate nucleus
PTg	pedunculotegmental areas
PBG	parabigeminal nucleus
PPT	pedunculopontine tegmental nucleus
PPN	pedunculopontine nucleus
3N	oculomotor nucleus
5N	the motor trigeminal nucleus
7N	the facial nucleus
6N	the abducens nucleus
10N	the dorsal motor nucleus of the vagus
12N	the hypoglossal nucleus
CIC	inferior colliculus
LDT	laterodorsal tegmental nucleus
DMT	dorsomedial tegmental nucleus
VTg	ventral tegmental nucleus
Me5	mesencephalic trigeminal nucleus
IPAC	posterior limb of the anterior commissure
LPO-PLH	lateral preoptic-hypothalamic continuum - peduncular part of the lateral hypothalamus

Ethics approval and consent to participate: Animal procedures were conducted under the National Institutes of Health’s Guide for Care and Use of Laboratory Animals and the Animal Research: Reporting in Vivo Experiments (ARRIVE) guiding principle. All procedures were in accordance with the regulations of the Animal Care and Ethics Committee of Western Sydney University (A14717), meaning experimental protocol A14717 covering all the performed experiments on mice, was approved by the Animal Care and Ethics Committee of Western Sydney University.
Consent for publication: Not applicable
Availability of data and materials: The data that support the findings of this study are available from Western Sydney University Research Library, but restrictions apply to the availability of these data, and so are not publicly available. Data are however available from the corresponding authors upon reasonable request and with permission of Western Sydney University.
Competing interests: The authors declare that they have no competing interests.
Funding: RG has been supported by a postgraduate scholarship by Western Sydney University.
Authors' contributions: RG and EG sacrificed the animals, collected tissue, prepared the sections, analyzed the data, prepared all the figures and tables, and drafted and finalized the manuscript. EG supervised and advised histological data processing and analysis. GM edited the manuscript draft, contributed to the discussion. LZ provided the transgenic animals, edited the manuscript draft, contributed to the discussion. All authors have reviewed the manuscript.
Acknowledgements: Not applicable

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Evaluation of eGFP expression in the ChAT-eGFP transgenic mouse brain

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Abstract

Figures

Background

Results

Forebrain regions that contain ChAT positive neurons

Telencephalon

Diencephalon

Midbrain and Hindbrain regions that contain ChAT positive neurons

Summary of anatomical areas with ChAT-eGFP and antibody expression

Discussion

Conclusion

Methods

Animals

Mapping

In situ – Allan Brain Institute

Confocal imaging

Abbreviations

Declarations

References

Additional Declarations

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Journal Publication

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