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Examination of In Vivo Effects of Semaphorins in the Nigrostriatal Pathway with Genetic Knockout Mice
1Chino
of Biology, Spelman College, Atlanta, GA; 2Department of Neurological Surgery, Emory University, Atlanta, GA
Abstract
Antibodies:
Rabbit anti-Tyrosine hydroxylase antibdoes were used to detect Tyrosine
hydroxylase-expressing neurons (PelFreeze). Mouse anti-Cre-recombinase
antibodies were used to detect Cre-recombinase (Chemicon).
Results
 A trend for more SNc Th+ neurons in Npn1loxP/loxP; ThCre+ brains
Crosses:
Npn1loxP/wt
X
Npn1loxP/loxP
5’
loxP
Npn1
loxP
3’
Cre
5’
Npn1loxP/wt
X
TH
Cre
X
3’
A
0
Cre+
Cre-
Cre ICC
Figure 9: Graph showing the average number
of Th positive neurons present in the DMN of
Th-Cre+ brains compared to Th-Cre- brains.
 A trend for more Th+ neurons in the paraventricular
hypothalamic nucleus (PVN) of Npn1loxP/loxP; Th-Cre+ brains
compared to controls
Figure 4: A) Substantia nigra
of a Th-Cre- brain and that of
a
Th-Cre+
brain
immunostained with anti-Th
antibodies.
600
500
400
300
B) Graph showing the
average number of Th
positive neurons present in
the SNc of Th-Cre+ brains
compared to Th-Cre- brains.
There is a trend for more
neurons per section in the Th-
200
100
0
Th-Cre+
TH-Cre-
Cre ICC
Cre+ brains.
(GFP)
P
loxP loxP
GFP
lacZ
3’
A
5’
Figure 5: A) Medial forebrain
bundle of an Npn1loxP/loxP;ThCre- brain.
B) Medial
forebrain bundle (mfb) of a
Npn1loxP/loxP;Th-Cre+
brain.
This bundle is more spread
out than that of the Th-Crebrain. The area of the mfb is
outlined for measurement.
Th-Cre
3’
B
B
loxP
Figure 2: A) Schematic showing how the animals were generated. B) Coronal brain sections
through the substantia nigra from (Npn1loxP/loxP;Th-Cre-) and (Npn1loxP/loxP;Th-Cre+) adult mice
were co-stained for Tyrosine hydroxylaze (TH in green) and Cre-recombinase (in red). Nuclei
were stained with Hoescht (blue). TH labeling is found in the cytoplasm of the neurons whereas
Cre-recombinase labeling is present in the nuclei as expected. In the (Npn1loxP/loxPxTh-Cre)
animals, many of the TH positive neurons are also posititive for Cre-recombinase.
Average Cell Number
Area of Hypothalamic Nucleus
200000
150000
100000
50000
0
Cre+
120
100
80
60
40
20
0
Cre+
Cre-
Cre ICC
Cre ICC
Figure 10: Graph comparing the average size
of PVN between Npn1loxP/loxP;Th-Cre+ and
Npn1loxP/loxP;Th-Cre- brains.
Cre-
Figure 11: Graph showing the average number
of Th positive neurons present in the PVN of ThCre+ brains compared to Th-Cre- brains.
Conclusions and Future Studies:
 Our preliminary results suggest that npn1 might participate in the number of Th
expressing neurons in SNc and PVN but not in the DMH.
 Npn1 also plays a subtle but significant role in restricting the spread of the mfb fibers.
 Future studies will include:
• ICC for Npn1 to determine the extent of Npn1 depletion caused by Cre expression;
• Increase number of animals examined for better statistics;
• Examine pathway at earlier developmental stages;
Genotyping and tissue preparation:
Mice were genotyped by PCR
immunocytochemistry.
and
Cre
expression
was
confirmed
• Examine potential behavioral effects of Th specific Npn1 deletion.
by
Resources
B
Area ± SDEV
Area of Medial Forebrain Bundle
A
50
Cre-
Figure 8: Graph comparing the average size of
DMN
between
Npn1loxP/loxP;Th-Cre+
and
Npn1loxP/loxP;Th-Cre- brains.
TH/Cre/Dapi
Cre
Npn1loxP/loxP
Th-Cre
GFP
50000
100
Npn1loxP/lox
ZBgeoloxP/wt
5’
loxP
100000
150
Cre ICC
 Significantly wider mfb in Npn1loxP/loxP; Th-Cre+ brains
Npn1loxP/loxP
Th-Cre
150000
Cre+
Npn1loxP/loxP
Th-Cre
Average Cell Number
0
A
Number of cells +STDV
Material and Methods
Npn 1 loxP/loxP;
Th-Cre-
200000
Cell Count of Substantia Nigra
Figure 1: A) Mesolimbic (blue) and nigrostriatal dopaminergic (green) tracts in human brain. B) The development
of the nigrostriatal pathway during embryogenesis in the mouse. Legand: Col – Colliculus; SN – Substantia Nigra;
Thal – Thalamus; LGE – Persisting Germinal Zone; Str – Straitum.
Npn 1 loxP/loxP; ThCre+
Area of Hypothalamic Nuclei
B
A
Figure 7: Sections through the
dorsomedial hypothalamic nucleus
of a Npn 1 loxP/loxP; Th-Cre+ and
a Npn 1 loxP/lox P; Th-Cre- brain
stained for Th. Note the collection
of Th labeled neurons on each side
of the ventricle.
Average Cell
Number/Section
Dopamine, norepinephrine and epinephrine are the main catecholamines that
play an important role as neurotransmitters in the central and peripheral nervous
systems. Dopamine controls behaviors such as voluntary movement, drug use and
abuse, motivation, reward and reinforcement. Tyrosine hydroxylase (TH), the rate
limiting enzyme in the synthesis pathway of these catecholamines, converts tyrosine into
3, 4-dihydroxyphenylanine, also known as levodopa (L-DOPA) (Gelman, 2003). L-DOPA
is converted to dopamine by an enzyme called L-amino acid decarboxylase (L-AADC).
Dopamine is then converted into norepinephrine and then into epinephrine. There are
many regions in the brain where catecholaminergic neurons are found (ex: A1 to A17 cell
nuclei).
Semaphorins are secreted or membrane-bound proteins that play a role in axon
guidance in several pathways in the brain. Semaphorins have cell surface receptors that
are bound to them with high affinity in these pathways. The known receptor for class 3
semaphorins (sema3A) is neuropilin 1 (npn1) and the co-receptor is plexin A1 (Fujisawa,
1997). Semaphorins bind to npn1 which is bound to its co-receptor, plexin A1. Plexin A1
then provides the signal through the cell membrane to the cell. Although we have
localized semaphorins in the nigrostriatal pathway, their role in this pathway has not yet
been identified.
To determine whether npn1 is necessary for the proper development of the
nigrostrital pathway we have crossed several strains of mice to generate a strain lacking
the npn1 gene only in the tyrosine hydroxylase expressing cells. We have then
compared the development of the nigrostriatal pathway and other dopaminergic
pathways between this mice lacking npn1 in TH expressing neurons and normal
controls.
 No difference in the number of Th+ neurons in the
dorsomedial hypothalamic nucleus (DMN) of Npn1loxP/loxP; ThCre+ brains compared to controls
Average Cell
Number/Section
Introduction
Immunocytochemistry:
Adult mouse brain tissue sections (N=6 (Npn1loxP/loxP;Th-Cre+) and N=14
(Npn1loxP/loxP;Th-Cre-))were collected and rinsed in PBS three times for 10 minutes
each. The tissues were incubated in hydrogen peroxide and 0.1% triton for 20
minutes to rid the cells of peroxidase and permeabilize the tissue, respectively. They
were washed in PBS again, in the manner stated above and incubated in a solution
of PBS and Normal Goat serum for blocking. The tissues were then washed with
PBS, and put in a primary antibody for overnight incubation in a room at 40oC. After,
the tissues were rinsed in PBS and incubated in a secondary antibody, a goat anti
rabbit IgG, rinsed and incubated in avidin-biotin complex. The tissues were put in a
solution of hydrogen peroxide and diaminobenzedene. They were washed in PBS
again in the manner stated above and the tissue sections were mounted on gelatin
coated slides.
Area ± Stdev
Parkinsons Disease (PD) is a neurodegenerative disorder that affects many people
worldwide. In patients with PD, there is a loss of dopaminergic neurons in the substantia
nigra pars compacta (SNc) located in the ventral midbrain. This loss of dopaminergic
neurons leads to a decreased level of dopamine. Our laboratory has preliminary data
showing that the semaphorins,a group of axon guidance molecules, are present in the
nigrostriatal pathway and therefore participate in its development. However, the exact
role of semaphorins in the nigrostriatal pathway has not yet been identified. Here, we use
genetic knockout mouse strains to examine the in vivo effects of semaphorins in the
development and establishment of the nigrostriatal pathway.
Area ± Stdev
1Department
S. Aneke, 2Elizabeth H. Jackson, 2Tisina Samaroo, 2Claire-Anne Gutekunst, 2Robert E. Gross
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
Figure 6: Graph comparing
the average mfb size between
Npn1loxP/loxP;Th-Cre+
and
Npn1loxP/loxP;Th-Crebrains.
The mfb fiber bundle is
significantly larger in the
Npn1loxP/loxP;Th-Cre+
compared to Npn1loxP/loxP;ThCre- brains. * indicates t-test
p=0.019.
*
Th-Cre+
Figure 3: A) Genotypes of P1 brains. The results show that the brains are npn-1 homozygous.
The brains that have a white band are Th-Cre+ ( 3 & 4), while those that do not (1 and 2) are
Th-Cre-. B) Cre staining of a brain section through the SNc of a Th-Cre+ brain. Cre is present
in the nuclei of many neurons in the SNc (outlined). Cre-recombinase is responsible for
excising the npn1 gene from the Npn1 loxP/loxP strain.
Th-CreCre ICC
This material is based upon work supported by the Howard Hughes Medical Institute
under Grant No. 52003727 and by the National Science Foundation Award # 0450303
(Subaward # I-66-606-63 to Emory University).
References
Gelman et. al. "Transgenic Mice Engineered to Target Cre/LoxP-Mediated DNA
Recombination Into Catecholaminergic Neurons". Technology Report 14 May 2007: 196202.
Lindeberg et.al. "Transgenic expression of Cre Recombinase From the Tyrosin
Hydroxylase Locus". Technology Report 16 March 2004: 67-73.
Fujisawa et.al, "Roles of a neuronal cell-Surfave Molecule, neuropilin, in nerve fiber
fasiculation and guidance". Cell & Tissue Research 11 June 1997: 465-470.
Novak et. al, "Article". Z/EG, a Double Reporter Mouse LIne That Expresses Enhanced
Green Fluorescent Protein Upon Cre-mediated Excision 28 September 2000: 147-155.