Midbrain

Midbrain is located between forebrain and hindbrain and is commonly segmented into tectum, aqueduct, tegmentum, basis pedunculi. The major part of tectum constitutes superior and interior colliculi (processing visual and auditory information, respectively). The cerebral aqueduct, which is the smallest ventricle in the brain linking 3rd and 4th ventricle, accommodates periaqueductal grey, dorsal raphe nucleus and several nuclei related to visual system. Tegmentum facilitates homeostasis and reflexes. The crucial tegmental nuclei include median raphe-, red nuclei (involved in motor coordination) and ventral tegmental area, the biggest dopamine production site. The basis pedunculi provides path to major nerve tracts, eg. corticospinal tract, internal capsule. It also contains affected in Parkinson disease substantia nigra, the dopamine production center. Developmentally, midbrain emerges from the second vesicle of neural tube and does not sub-divide further, in comparison to other brain regions.

The transcriptome analysis shows that 83% (n=16240) of all human protein-coding genes (n=19670) 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 11762 of all mouse one-to-one orthologues (n=15160) are expressed in the mouse midbrain and that 12819 of all pig orthologues (n=14656) are expressed in the pig midbrain.

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

The transcriptome analysis shows that 83% (n=16240) of all human protein-coding genes (n=19670) 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 11762 of all mouse one-to-one orthologues (n=15160) are expressed in the mouse midbrain and that 12819 of all pig orthologues (n=14656) 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 8 out of those genes show highest expression levels in midbrain in all three species.

Sub regions of the midbrain

Raphe nucleus

Raphe nuclei are clusters of nuclei in the brain stem, containing
serotonergic neurons. The more posterior nuclei reside in pons and medulla and project to spinal cord and cortical areas. The parts residing in the midbrain, dorsal and median raphe nucleus (MRN) are considered to be implicated in the depression. The MRN outputs target the Hippocampus and are involved in the long-term memory formation. The dorsal raphe nucleus projects to amygdala, cerebral cortex, caudate, putamen and olfactory bulb.


TPH2 - Dorsal raphe

TPH2 - Caudate nucleus


SLC6A4 - Dorsal raphe

SLC6A4 - Caudate nucleus


SNCG - Median raphe

PAK1 - Dorsal raphe

Ventral tegmental area

Ventral tegmental area (VTA) is the largest center for production of dopamine, and together with Substantia Nigra, origin of the major dopamine pathways: 1) mesolimbic (projecting to nucleus accumbens and olfactory tubercle); 2) mesocortical (delivering dopamine to the prefrontal cortex). VTA outputs are also targeting Hippocampal formation, Amygdala and Olfactory bulb. Most of the neuronal connections are reciprocal. The VTA is implicated in the reward system (addiction), cognition and psychiatric disorders.


TH - Ventral tegmental area

Substantia Nigra

Substantia Nigra is divided into two parts: 1) pars compacta (SNc) projects to the caudate and putamen (dopaminergic niagrostratal pathway); 2) pars reticulata (SNpr) receives inputs from basal ganglia (direct and indirect pathway) and conveys to other brain areas, including thalamus and superior colliculus. SN is involved in the reward system and motor control. The name substantia nigra (latin for black substance) comes from the darker color of the residing dopaminergic cells, containing pigment neuromelanin. Loss of dopaminergic neurons is implicated in Parkinson's Disease.


SNCG - Substantia nigra

DRD2 - Substantia nigra


SLC6A3 - Substantia nigra

Regionally elevated expression in human (Substantia Nigra)

The transcriptome analysis shows that 83% (n=16240) of all human proteins (n=19670) are expressed in the midbrain. 56 genes show an elevated expression level in midbrain compared to other regions of the brain.

  • 14 regionally enriched genes
  • 56 regionally elevated genes in total
  • 34 of the midbrain elevated genes are elevated in other tissues than the brain.
  • 22 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 14
Group enriched 31
Region enhanced 11
Elevated 56

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 among the 14 enriched genes 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
SLC6A3 Solute carrier family 6 member 3 Membrane 47
PITX3 Paired like homeodomain 3 Intracellular 18
MIA Melanoma inhibitory activity Secreted 8
FGFBP2 Fibroblast growth factor binding protein 2 Secreted 6
FOXA1 Forkhead box A1 Intracellular 6
IL17B Interleukin 17B Secreted 6
CNTF Ciliary neurotrophic factor Intracellular 5
HCAR3 Hydroxycarboxylic acid receptor 3 Membrane 5
PAX5 Paired box 5 Intracellular 5
DHH Desert hedgehog Secreted 4
IL1R2 Interleukin 1 receptor type 2 Membrane,Secreted 4
MPZ Myelin protein zero Intracellular,Membrane 4


SLC6A3 - Substantia nigra

SLC6A3 - Caudate nucleus

Regionally elevated expression in mouse midbrain

The whole of 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=11762) of all mouse one-to-one human orthologue genes (n=15160) are expressed in the midbrain and 44 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 28
Region enhanced 8
Elevated 44


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
SLC6A4 Solute carrier family 6 member 4 Membrane 5
TAL1 TAL bHLH transcription factor 1, erythroid differentiation factor Intracellular 5
TFAP2D Transcription factor AP-2 delta Intracellular 4


PAX5

SLC6A4

Regionally elevated expression in pig midbrain

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

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

The transcriptome analysis shows that 65% (n=12819) of all pig one-to-one human orthologue genes (n=14656) 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 24
Group enriched 49
Region enhanced 33
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 139
MPZ Myelin protein zero Intracellular,Membrane 57
PRX Periaxin Intracellular 21
FERD3L Fer3 like bHLH transcription factor Intracellular 19
TH Tyrosine hydroxylase Intracellular 19
INS Insulin Secreted 13
SSPO SCO-spondin Secreted 13
FXYD2 FXYD domain containing ion transport regulator 2 Membrane 9
SPDEF SAM pointed domain containing ETS transcription factor Intracellular 8
ASB4 Ankyrin repeat and SOCS box containing 4 Intracellular 7
FOXA2 Forkhead box A2 Intracellular 7
TFAP2D Transcription factor AP-2 delta Intracellular 7

Extended human midbrain tissue section

The standard setup in the Tissue Atlas, that profiles the human tissues, is based on Tissue Micro array technique, saving valuable tissue material as well as reagents but still provide a good tissue representation for protein profiling. Cerebral cortex, caudate nucleus, hippocampus and cerebellum are the four brain regions in the standard human brain profiling. However, when literature suggests a function related to either Substantia nigra or Dorsal raphe, or when RNA expression data suggests selective expression in those two regions, special tissue material have been added to the protein profiling. So far, only 4 different genes have been analyzed on the extended material.

Table 7. The following 4 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.
SLC6A3 Solute carrier family 6 member 3 Substantia nigra Strong cytoplasmic staining in neurons.
SLC6A3 Solute carrier family 6 member 3 Dorsal raphe Rare projection positivity.
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.