Midbrain Anatomical divisions Regionally elevated protein expression in human Regionally elevated protein expression in mouse Regionally elevated protein expression in pig Extended information

Midbrain

Summary

The midbrain is located between forebrain and hindbrain and is commonly segmented into tectum, aqueduct, tegmentum, and basis pedunculi. Developmentally, midbrain emerges from the second vesicle of the neural tube and does not sub-divide further, in comparison to other brain regions. The midbrain is involved in functions such as the processing of visual and auditory information, facilitating homeostatic reflexes and motor coordination.

The transcriptome analysis shows that 72% (n=14409) of all human protein-coding genes (n=20090) are expressed in the human midbrain (represented by RNA expression in substantia nigra). Human one-to-one orthologues were investigated in pig and mouse brain, suggesting that 12147 of all mouse one-to-one orthologues (n=16320) are expressed in the mouse midbrain and that 13335 of all pig orthologues (n=15829) are expressed in the pig midbrain.

Gene classification based on regional RNA expression in human, pig and mouse midbrain defines 37 genes as midbrain enriched in either of the three species, and 13 out of those genes show highest expression levels in midbrain in all three species.

Figure 1. Schematic drawing of the human brain, indicating the location of midbrain from a sagittal view.

Anatomical divisions

The midbrain grey matter can be divided into the pretectal region, tegmentum, and tectum. The pretectal area, situated close to the forebrain, is part of the mediation of pupal reflexes and is indicated in the induction of REM sleep. Within the tegmentum resides periaqueductal grey (anterior, dorsal, and lateral part) which envelops the cerebral aqueduct and is involved in the visual system as well as implicated in pain suppression. Other important nuclei in the midbrain are the dorsal raphe and median raphe nucleus. The dorsal raphe nucleus projects to amygdala, cerebral cortex, caudate, putamen and olfactory bulb. While the median raphe nucleus outputs target the hippocampus and are involved in long-term memory formation. Both nuclei are implicated in depression.

TPH2 - Dorsal raphe
SLC6A4 - Dorsal raphe
PAK1 - Dorsal raphe
SNCG - Median raphe

Moreover, in tegmentum is the ventral tegmental area which is the largest centre for production of dopamine in the brain. It has outputs targeting several brain regions e.g., the hippocampal formation, amygdala, and olfactory bulb. It is implicated in functions concerning the reward system and addiction, cognition, and psychiatric disorders. The cuneiform nucleus and the pedunculopontine tegmental nucleus are part of the mesencephalic locomotor region and are in the dorsolateral portion of tegmentum. Centrally in the tegmentum is the large structure the red nucleus that is involved in motor coordination. Another area of tegmentum involved in motor functions as well as the reward system is the substantia nigra. The substantia nigra is divided into two parts: 1) pars compacta (that projects to the caudate nucleus and putamen via the dopaminergic nigrostriatal pathway); 2) pars reticulata (that receives inputs from basal ganglia, via the direct and indirect pathway). It is a large group of pigmented dopaminergic neurons that together with globus pallidus, caudate nucleus, and putamen form the striatum. Additionally, the substantia nigra is implicated in Parkinson’s disease.

TH - Ventral tegmental area
SNCG - Substantia nigra
DRD2 - Substantia nigra
SLC6A3 - Substantia nigra

Tectum is the dorsal part of the midbrain. A major part of tectum is comprised of the superior colliculus and inferior colliculus which, respectively, play a role in the processing of visual and auditory information

Regionally elevated protein expression in human

The transcriptome analysis shows that 72% (n=14409) of all human proteins (n=20090) are expressed in the midbrain. 80 genes show an elevated expression level in midbrain compared to other regions of the brain.

18 regionally enriched genes
80 regionally elevated genes in total
30 of the midbrain elevated genes are elevated in other tissues than the brain.
50 of the midbrain elevated genes are elevated in the brain.

Table 1: Schematic drawing of the human brain, indicating the location of midbrain from a coronal perspective or sagittal view.

Specificity	Number of Human elevated genes
Region enriched	18
Group enriched	37
Region enhanced	25
Elevated	80

Elevated expression in midbrain compared to other brain regions is divided into three different categories; regionally enriched (at least four-fold higher mRNA levels in midbrain compared to all other regions), group enriched (at least four-fold higher mRNA levels in a group of 2-5 regions) and regionally enhanced (at least four-fold higher mRNA levels in midbrain compared to the average of all regions), The number of genes in the individual category is shown in Table 1. In Table 2, the 12 genes with the highest level of regional specificity amongof the 18 genes with high level of regional specificity are listed.

Table 2. The 12 genes with the highest level of enriched expression in human midbrain. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
GPR151	G protein-coupled receptor 151	Membrane	40
SLC6A3	Solute carrier family 6 member 3	Membrane	26
SLC18A1	Solute carrier family 18 member A1	Membrane	19
PITX3	Paired like homeodomain 3	Intracellular	14
SRD5A2	Steroid 5 alpha-reductase 2	Membrane	11
IGHV4-4	Immunoglobulin heavy variable 4-4	Secreted	10
COL6A5	Collagen type VI alpha 5 chain	Intracellular,Secreted	7
NTSR1	Neurotensin receptor 1	Membrane	7
SDC1	Syndecan 1	Intracellular,Membrane,Secreted	7
CLEC3A	C-type lectin domain family 3 member A	Secreted	6
EN1	Engrailed homeobox 1	Intracellular	6
TFF3	Trefoil factor 3	Intracellular,Secreted	6

SLC6A3 - Substantia nigra
SLC6A3 - Caudate nucleus

Regionally elevated protein expression in mouse

The whole mouse midbrain was dissected and used for this analysis, mean value of the 2 male and 2 female mice is used as representative of the midbrain.

The transcriptome analysis shows that 60% (n=12147) of all mouse one-to-one human orthologue genes (n=16320) are expressed in the midbrain and 45 genes show an elevated expression in midbrain compared to other regions of the brain.

Table 3: Number of genes within the different categories of regionally elevated expression, in mouse midbrain

Specificity	Number of Mouse elevated genes
Region enriched	8
Group enriched	27
Region enhanced	10
Elevated	45

Figure 2. Schematic drawing of the mouse brain, indicating the location of midbrain from a sagittal view.

Table 4: The 8 genes with the highest level of enriched expression in mouse midbrain. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Gene	Description	Predicted location	RS-score
PAX7	Paired box 7	Intracellular	17
PAX5	Paired box 5	Intracellular	11
GATA3	GATA binding protein 3	Intracellular	6
POU4F2	POU class 4 homeobox 2	Intracellular	6
EN1	Engrailed homeobox 1	Intracellular	5
TAL1	TAL bHLH transcription factor 1, erythroid differentiation factor	Intracellular	5
SLC6A4	Solute carrier family 6 member 4	Membrane	4
TFAP2D	Transcription factor AP-2 delta	Intracellular	4

PAX5
SLC6A4

Regionally elevated protein expression in pig

Figure 3. Schematic drawing of the pig brain, indicating the location of midbrain from a sagittal view.

The substantia nigra, superior colliculus and periaqueductal gray were dissected out as separate areas of the midbrain and analyzed separately from the remaining midbrain area. The highest expression value of the 4 midbrain samples was used as representative for midbrain in the regional expression comparison.

The transcriptome analysis shows that 66% (n=13335) of all pig one-to-one human orthologue genes (n=15829) are expressed in the midbrain. 106 genes show an elevated expression in midbrain compared to other regions of the brain.

Table 5: Number of genes within the different categories of regionally elevated expression, in pig midbrain

Specificity	Number of Pig elevated genes
Region enriched	18
Group enriched	37
Region enhanced	51
Elevated	106

Table 6: The 12 genes with the highest level of enriched expression in pig midbrain. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
SLC6A3	Solute carrier family 6 member 3	Membrane	152
TH	Tyrosine hydroxylase	Intracellular	20
FERD3L	Fer3 like bHLH transcription factor	Intracellular	16
INS	Insulin	Secreted	13
ASB4	Ankyrin repeat and SOCS box containing 4	Intracellular	10
FOXA2	Forkhead box A2	Intracellular	9
SPDEF	SAM pointed domain containing ETS transcription factor	Intracellular	7
TTC6	Tetratricopeptide repeat domain 6	Intracellular	7
ALDH1A1	Aldehyde dehydrogenase 1 family member A1	Intracellular	6
CHRNA6	Cholinergic receptor nicotinic alpha 6 subunit	Intracellular,Membrane	6
EN1	Engrailed homeobox 1	Intracellular	6
FOXA1	Forkhead box A1	Intracellular	6

Extended information

Table 7. The following 5 genes have been analyzed using extended midbrain samples.

Gene	Gene description	Tissue	Staining pattern
DBH	Dopamine beta-hydroxylase	Dorsal raphe	Strong synaptic and projection staining, membranous staining in neurons.
PRPH	Peripherin	Dorsal raphe	Cytoplasmic positivity in a subset of neurons.
SLC6A3	Solute carrier family 6 member 3	Dorsal raphe	Rare projection positivity.
SLC6A3	Solute carrier family 6 member 3	Substantia nigra	Strong cytoplasmic staining in neurons.
SLC6A4	Solute carrier family 6 member 4	Dorsal raphe	Strong membrane staining in a few neurons. Synaptic and projection staining.
TPH2	Tryptophan hydroxylase 2	Dorsal raphe	Strong neuronal staining.

Substantia nigra and Parkinsson's disease

This movie shows 3D light sheet microscopy scans of the neurons that are affected by Parkinson´s disease, the dopamine producing neurons located in the substantia nigra. Interview: Prof Per Svenningsson, Karolinska Institutet.