Ok so anyone who wants to reproduce my problem will need 2 files.
1. A dummy training file (a single annotated pharmacology paper)
2. The same paper but raw. In other words the file before being annotated.
3. As an extra i'm including the model i just trained on this paper.
What i'm getting back from the trained model when i feed it with the
tokenised sentences of the original raw text is:
/
("hydroxysaclofen") ("muscimol" "hydroxysaclofen") ("Hydroxysaclofen")/
This is really not good!!! Only 3 sentences mentioned drugs?
No multi-word drugs even though they exist in training text.
_File description:_
* PROPER-TRAIN-DATA.txt ---> as the name implies...one sentence per
line, spaces in sgml tags, empty line at the end
* my-2NER-short.bin ----> the model i just trained using
PROPER-TRAIN-DATA.txt
* 48147.nex.txt ----> the original raw paper.
It takes less than 5 seconds to train from such a small file and even
less to run the trained NER so try it out and let me know how get on...
Regards,
Dimitris
On 08/02/12 19:09, Jörn Kottmann wrote:
I see the following issues:
- Multiple sentences in a line
- You data is not tokenized
- Adaptive data is not cleared
You can use our sentence detector to split
your paragraphs. If you know your document
boundaries you should write an empty line to
that file to clear the adaptive data. If you cannot
do that write an empty line after every sentence.
Do you use our command line tools for training?
Jörn
On 02/08/2012 06:46 PM, Jim - FooBar(); wrote:
Would it be possible for you to show us a sample of your training data?
Maybe one paper.
Absolutely here you go....a sample has been attached...Let me know if
you want more but i can assure you that since the sgml tags are
generated automatically (with regex replacement) they are all of the
same format...
Jim
p.s: fire up your favourite editor press ctrl+f and search for
"<START" just to see locate them easily!
On 08/02/12 17:09, Joern Kottmann wrote:
On Wed, Feb 8, 2012 at 5:56 PM, Jim -
FooBar();<jimpil1...@gmail.com>wrote:
aaa ok i see what you mean...but then again if it recognised it as
a mere
token it would not throw "IncompatibleFormat" exceptions but rather
skip it
as a token that is not of interest wouldn't it? I don't have any
patches to
send you, i just think that not including spaces in the sgml tag is
a more
wise approach...Unless of course you're extracting the sgml tags via
regex...The truth is i've not looked at the source but i would
expect you
to use some sort of xml-ish means to extract the sgml tags. If your
parser
is using regex then i'm sure you have your reasons for including the
spaces. But anyway, this is a very small problem for me cos i can
indeed
sort it manually...My big problem still remains!!!
The code splits the input string by line and then by white space.
Then the
individual parts either
match our start and end tags or not.
Anyway I'll stop bugging you...the fact that you tried to help
means a lot
and certainly if i sort everything out i'll post what the problem
was for
future users...
We are also interested why it does not work for you, we usually use
this
kind of experience to
improve OpenNLP.
Would it be possible for you to show us a sample of your training data?
Maybe one paper.
Jörn
The effect of GABA receptor ligands in experimental spina bifida occulta.
Background.
The pathophysiology behind spina bifida and other neural tube defects (NTDs) is
unclear.
Folic acid is one variable, but other factors remain. Studies suggest that
substances active at the GABA receptor may produce NTDs.
To test this hypothesis pregnant rats were exposed to either the GABA a agonist
muscimol (1, 2 or 4 mg/kg), the GABA a antagonist bicuculline (.5, 1, or 2
mg/kg), the GABA b agonist <START:drug> Baclofen <END> (15, 30, 60 mg/kg), or
the GABA b antagonist hydroxysaclofen (1, 3, or 5 mg/kg) during neural tube
formation.
Normal saline was used as a control and <START:drug> Valproic Acid <END> (600
mg/kg) as a positive control.
The embryos were analyzed for the presence of a spina bifida like NTD.
Results.
After drug administration the pregnancies were allowed to proceed to the 21 st
day of gestation.
Then embryos were removed and skeletons staining and cleared.
Vertebral arch closure was measured.
Results indicate that the GABAa receptor agonist muscimol, the GABAa receptor
antagonist bicuculline, and the GABAb agonist <START:drug> Baclofen <END>
produced NTDs characterized by widening of the vertebral arch.
Oppositely the GABAb antagonist hydroxysaclofen produced narrowing of the
vertebral arches.
Conclusions.
The findings indicate that GABA a or b ligands are capable of altering neural
formation.
GABA may play a greater than appreciated role in neural tube formation and may
be important in NTDs.
The narrowing of the vertebral arch produced by the GABA b antagonist
hydroxysalcofen suggests that GABA b receptor may play an undefined role in
neural tube closure that differs from the GABA a receptor.
Background.
Neural tube defects (NTDs) are major malformations of the central nervous
system (CNS) due to a defect in the covering of the CNS.
They are among the most prevalent of congenital malformations.
NTDs are second only to congenital heart defects as a cause of perinatal
mortality due to birth defect and range in incidence from 0.5 to 12 per 1000
live births, depending on the country, accounting for 400,000 births world-wide
annually.
Factors that predispose individuals to NTDs are numerous.
While <START:drug> Folic Acid <END> deficiency and altered <START:drug> Folic
Acid <END> metabolism have received widespread attention, other contributors
are also important.
Socio-economic status, genetic factors, maternal illness and maternal drug
exposure are important contributors to the risk of NTDs.
Drug models of NTDs are valued because the drug's action provides a possible
explanation of the pathophysiology of NTDs.
Valproic acid (VA) is a well-known teratogen in both animals and humans, with a
5-fold occurrence of spina bifida (SB) in pregnant women exposed to the drug [
1 ].
The mechanism by which VA produces SB is unknown but inhibition of <START:drug>
Folic Acid <END> metabolism is one hypothesis [ 2 , 3 ].
Some investigators have brought the <START:drug> Folic Acid <END> hypothesis
into question by demonstrating that <START:drug> Folic Acid <END>
supplementation has no effect on VA exposed embryos in-vitro [ 4 , 5 ].
These studies suggest that another mechanism may be responsible for the
production of SB by VA.
Other possible mechanisms include alteration of neuronal membrane conductance,
sodium channel blockade or altered neuronal <START:drug> Calcium <END>
metabolism [ 6 ].
VA is also known to allow GABA, the chief inhibitory neurotransmitter of the
CNS, to accumulate in tissues.
The role of GABA as a potential site for teratogen activity for VA and other
teratogens has been little explored.
Preliminary reports have linked ligands active at the GABA receptor with SB or
other NTDs, including benzodiazepines, alcohol, and <START:drug> Zinc <END> [ 7
- 11 ].
Preliminary work from this laboratory has shown the GABAa receptor agonist
muscimol and the GABAb agonist <START:drug> Baclofen <END> produce both SB and
the Arnold-Chiari malformation, commonly associated with SB [ 12 - 14 ].
VA appears to produce most of its anticonvulsant effects by increasing levels
of GABA in the CNS, presumably by inhibiting the enzyme GABA-transaminase
(GABA-T).
There is other evidence to suggest that substances which alter the function of
the GABAergic system may contribute to the formation of NTDs.
Alcohol has been associated with NTDs [ 11 ] and is known to enhance the
functioning of GABA.
Benzodiazepines (BDZs), which enhance the activity of the GABA receptor, also
enhance the teratogenic effects of VA in humans [ 10 ].
Chlordiazepoxide, another BDZ, has been shown to produce NTDs in the hamster [
11 ].
Further support for the contention that VA may produce NTDs by way of GABA
activity include the following evidence.
One hypothesis of VAs mechanism of action holds that it alters intracelluar pH.
This may be the case as GABA can increase intracellular proton levels by
intensifying bicarbonate ion conductance through a GABA-gated channel [ 15 ]
which may act as a "developmental handshake" and regulate neuronal
differentiation [ 16 ].
The chloride channel, an integral part of the GABA a receptor, has been
implicated in embryonic development [ 17 ].
GABA receptors are first seen at the time the neural tube formation [ 18 ].
Binding sites to GABA agonists and antagonists and the expression of GABA
receptor mRNAs are seen starting at 4 days of development and peak at 10â15
days, corresponding to the time of neural tube formation [ 19 ].
Many neurotransmitters, including GABA, are growth factor candidates for the
CNS [ 20 - 22 ].
Based on the above information we have undertaken this study to systematically
examine the effects of GABA agonists and antagonists at both the GABA a and b
receptor and the role they may play in SB.
We used an established model of SB in the rat to test the hypothesis using VA
as a known standard.
This model of SB uses the width of the vertebral arch as an indicator of neural
tube closure.
While this model does not produce a specific SB lesion the widening of the
vertebral arch provides a model that resembles human SB in terms of
accompanying defects [ 12 - 14 , 23 ] and response to folate and other drugs [
6 , 11 , 24 , 25 ] and meets the formal definition of a neural tube defect: any
defect in the covering of the central nervous system.
We report here the effects of GABA a and b receptor agonists and antagonists
administered to rats at 10 days gestation, the period of neural tube formation.
Results and Discussion
A total of 1156 embryos from 123 litters were examined. Measurements from the
embryos were averaged for each litter and the litter was used as the unit of
analysis.
All measurements were made in a blind manner.
ANOVA revealed a significant effect of drug treatment of the average vertebral
arch distance (F(13, 109)=7.70, p < .0001).
The Bonferroni test was used for follow-up comparisons and the comparisons to
the normal saline group are given below and in Table 1 and in Figure 1 where
the results are presented graphically.
Occasionally other defects were noted in the embryos, chiefly fused ribs, these
defects were not appreciable and did not impact the vertebral arch analysis.
There is no statistically significant relationship between mean vertebral arch
distance and mean littler size.
Effect of GABA receptor ligands on vertebral arch distance.
Mean vertebral arch distance for drug groups. Error bars represent standard
deviations.
Exact probability differences from normal saline are given above error bars.
See text for comments.
Valproic Acid produced a significant widening of the vertebral arch in a manner
consistent with previous reports (p < .0005) [ 6 , 24 ].
The GABAa agonist muscimol produced a significant widening of the vertebral
arch at all three doses tested (1, 2, 4 mg/kg) (p=.06 for 1 mg/kg, p < .05 for
2 & 4 mg/kg).
The GABAa antagonist bicuculline also widened vertebral arch distance at the
0.5 and 1 mg/kg doses (p < .05), but not at the 2 mg/kg dose.
The GABAb agonist <START:drug> Baclofen <END> produced significant widening of
the vertebral arch at 30 mg/kg (p < .05) but not at 15 or 60 mg/kg.
The GABAb antagonist <START:drug> hydroxysaclofen <END> produced a significant
narrowing of the vertebral arch at all three doses tested (p=.09 for 1 mg/kg,
p=.001 for 3 mg/kg and p=.05 for 5 mg/kg).
This study indicates that substances active at either the GABA a or b receptors
have teratogenic potential.
The most striking feature of the drug effects is the differential effect of
antagonizing the GABAb receptor with <START:drug> hydroxysaclofen <END>.
While the GABAa agonist <START:drug> muscimol <END> and antagonist bicucullin
widened the vertebral arch, as did the GABAb agonist baclofen, the GABAb
antagonist <START:drug> hydroxysaclofen <END> narrowed the vertebral arch.
Narrowing of the vertebral arch was unexpected but has been previously
reported.
Previous work in this laboratory has demonstrated narrowing of the vertebral
arch when <START:drug> Zinc <END> is co-administered with <START:drug> Baclofen
<END> or muscimol [ 13 ].
It is unclear what the structural or functional consequence of vertebral arch
narrowing is.
Work in this laboratory has shown lags in neuromuscular development associated
with excessive <START:drug> Zinc <END> exposure during neural tube formation
and, possibly, accompanying narrowing of the vertebral arches [ 26 ].
Another curious finding of this study is that widening of the vertebral arch
occurs with either the GABAa agonist muscimol or the antagonist bicuculline.
In classic pharmacology it would be expected that the effects would be
opposite.
However, in this instance it may be that any disruption of the normal function
of the GABAa receptor and its integral chloride channel result in widening of
the vertebral arch.
On the other hand the GABAb receptor is not directly linked to a chloride
channel exerting its effect via second messenger systems.
This functional difference may allow for a classic agonist-antagonist drug
effect during neural tube formation.
One potential confound of this study is the role of developmental delays.
Animals exposed to valproate and presumably suffering from widened vertebral
arches can show weight differences well into post-natal life.
However, behavioral differences persist even when weight differences no longer
exist [ 1 ].
However, we are unaware of any studies directly examining the spinal columns of
animals well into post-natal life, therefore it is unclear if the widening of
the vertebral arch seen at 21 days persists.
Another limitation to this study is the small sample sizes for some groups and
the administration of drugs to only one day.
These results need to be replicated using larger sample sizes and administering
the drugs at other times during gestation.
It is possible that these drugs may disrupt neural tube formation if given
outside of the classic time frame for neural tube formation.
GABA is a well-documented neurotrophic agent involved in brain development [ 27
- 30 ].
Most of the work done on the effects of GABA and neural development has been
done on embryos and embryonic tissue well past the neural tube stage [ 27 , 31
, 32 ].
However, there is evidence of glutamic acid decarboxylase (GAD) and GABA
receptor expression about the time of neural tube formation [ 27 ].
Given that GABA is important to neural development, and the early developmental
time frame of the GABA system, it is logical that agents active at the GABA
receptor (ethanol, BDZs) can have adverse consequences on CNS development.
Nearly all of the substances examined are GABAa receptor ligands. GABAb
receptor effects have been little studied and the role of the GABAb receptor in
neural development is little known and should be more thoroughly investigated.
Conclusions.
GABA may play an important role in neural tube formation and the production of
neural tube defects.
Substances active at the GABA a or b receptor may be potentially teratogenic.
In particular, the findings indicate that GABA a or b ligands are capable of
altering neural formation.
GABA may play a greater than appreciated role in neural tube formation and may
be important in neural tube defects.
The narrowing of the vertebral arch produced by the GABA b antagonist
hydroxysalcofen suggests that GABA b receptor may play an important, but
undefined role in neural tube closure that differs from the GABA a receptor.
Materials and Methods.
Female Long-Evans rats 120 days of age were housed with ad-lib access to food
and rat chow (Purina, Brentwood, MO) under 12:12 light:dark conditions.
The females were mated with males of the same age and strain overnight with the
observation of a copulatory plug as evidence of mating and counted as day 0 of
pregnancy.
The females were then separated and housed singly.
At 10 days of gestation the females were treated with one drug as described
below.
Ten days of gestation corresponds with neural tube formation in the rat.
All drugs and chemicals were obtained from Sigma Chemical, St, Louis, MO.
Doses were empirically determined with pilot studies.
This study was reviewed and approved by the Institutional Animal Care and Use
Committee of the University of Nebraska at Kearney.
Reference groups.
Normal saline intraperitoneally (IP) 0.9 cc to establish baseline.
<START:drug> Valproic Acid <END> subcutaneously (SC) 1200 mg/kg (600 mg/cc) in
two divided doses over 8 hours.
Previous studies have shown this dose and route to reliably produce SB in the
rat [ 24 ].
This group provided a positive control to which the teratogenic activity of
other substances could be compared.
GABAa test groups.
<START:drug> Muscimol <END> IP at 1, 2, or 4 mg/kg (1 mg/cc). Muscimol is a
well-documented specific and potent GABAa agonist [ 33 ].
<START:drug> Bicuculline <END> methiodide IP at 0.5, 1 or 2 mg/kg (1 mg/cc).
<START:drug> Bicuculline <END> is a well-documented specific and potent GABAa
antagonist [ 33 ].
GABAb test groups.
Baclofen IP at 15, 30, or 60 mg/kg (9 mg/cc as aqueous suspension).
Baclofen is a well-documented and specific GABAb agonist [ 34 ].
Hydroxysaclofen IP at 1, 3, or 5 mg/kg (1 mg/cc).
<START:drug> Hydroxysaclofen <END> is a well-documented specific and potent
GABAb antagonist [ 34 ].
Embryo studies.
After injection gestation was allowed to otherwise progress as normal.
At 21 days gestation the females were euthenized with chloroform, the abdomen
opened and the uterus and uterine contents removed.
Fetuses were removed and had their abdomens opened to allow for the penetration
of 10% 0.1 M phosphate buffered formalin in which they were immersed.
After three days fixation the fetuses were stained for bone and cartilage as
described previously [ 24 ].
Briefly, the fetuses were eviscerated and skinned. Cartilage was stained with
alcian blue followed by bone staining with alizarin red.
The fetuses were cleared in KOH and graded concentrations of glycerol.
After clearing the fetuses were inspected with a dissecting microscope and the
width of the vertebral arch gap was measured with an eyepiece micrometer from
T-9 to S-4.
Previous work has shown that these vertebrae are the most clearly visible and
the most frequently effected [ 6 , 24 ].
For the sake of clarity vertebral arch distances from T-9 to S-4 were averaged
for the each embryo.
For analysis vertebral arch distance was averaged for each fetus and then for
the litter.
Data was analyzed with Analysis of Variance with follow-up statistics
(Bonferroni test) using Statview 5.0 for the Macintosh.
The effect of GABA receptor ligands in experimental spina bifida occulta
Background
The pathophysiology behind spina bifida and other neural tube defects (NTDs) is
unclear. Folic acid is one variable, but other factors remain. Studies suggest
that substances active at the GABA receptor may produce NTDs. To test this
hypothesis pregnant rats were exposed to either the GABA a agonist muscimol (1,
2 or 4 mg/kg), the GABA a antagonist bicuculline (.5, 1, or 2 mg/kg), the GABA
b agonist baclofen (15, 30, 60 mg/kg), or the GABA b antagonist hydroxysaclofen
(1, 3, or 5 mg/kg) during neural tube formation. Normal saline was used as a
control and valproic acid (600 mg/kg) as a positive control. The embryos were
analyzed for the presence of a spina bifida like NTD.
Results
After drug administration the pregnancies were allowed to proceed to the 21 st
day of gestation. Then embryos were removed and skeletons staining and cleared.
Vertebral arch closure was measured. Results indicate that the GABAa receptor
agonist muscimol, the GABAa receptor antagonist bicuculline, and the GABAb
agonist baclofen produced NTDs characterized by widening of the vertebral arch.
Oppositely the GABAb antagonist hydroxysaclofen produced narrowing of the
vertebral arches.
Conclusions
The findings indicate that GABA a or b ligands are capable of altering neural
formation. GABA may play a greater than appreciated role in neural tube
formation and may be important in NTDs. The narrowing of the vertebral arch
produced by the GABA b antagonist hydroxysalcofen suggests that GABA b receptor
may play an undefined role in neural tube closure that differs from the GABA a
receptor.
Background
Neural tube defects (NTDs) are major malformations of the central nervous
system (CNS) due to a defect in the covering of the CNS. They are among the
most prevalent of congenital malformations. NTDs are second only to congenital
heart defects as a cause of perinatal mortality due to birth defect and range
in incidence from 0.5 to 12 per 1000 live births, depending on the country,
accounting for 400,000 births world-wide annually.
Factors that predispose individuals to NTDs are numerous. While folic acid
deficiency and altered folic acid metabolism have received widespread
attention, other contributors are also important. Socio-economic status,
genetic factors, maternal illness and maternal drug exposure are important
contributors to the risk of NTDs.
Drug models of NTDs are valued because the drug's action provides a possible
explanation of the pathophysiology of NTDs. Valproic acid (VA) is a well-known
teratogen in both animals and humans, with a 5-fold occurrence of spina bifida
(SB) in pregnant women exposed to the drug [ 1 ]. The mechanism by which VA
produces SB is unknown but inhibition of folic acid metabolism is one
hypothesis [ 2 , 3 ]. Some investigators have brought the folic acid hypothesis
into question by demonstrating that folic acid supplementation has no effect on
VA exposed embryos in-vitro [ 4 , 5 ]. These studies suggest that another
mechanism may be responsible for the production of SB by VA. Other possible
mechanisms include alteration of neuronal membrane conductance, sodium channel
blockade or altered neuronal calcium metabolism [ 6 ]. VA is also known to
allow GABA, the chief inhibitory neurotransmitter of the CNS, to accumulate in
tissues.
The role of GABA as a potential site for teratogen activity for VA and other
teratogens has been little explored. Preliminary reports have linked ligands
active at the GABA receptor with SB or other NTDs, including benzodiazepines,
alcohol, and zinc [ 7 - 11 ]. Preliminary work from this laboratory has shown
the GABAa receptor agonist muscimol and the GABAb agonist baclofen produce both
SB and the Arnold-Chiari malformation, commonly associated with SB [ 12 - 14 ].
VA appears to produce most of its anticonvulsant effects by increasing levels
of GABA in the CNS, presumably by inhibiting the enzyme GABA-transaminase
(GABA-T). There is other evidence to suggest that substances which alter the
function of the GABAergic system may contribute to the formation of NTDs.
Alcohol has been associated with NTDs [ 11 ] and is known to enhance the
functioning of GABA. Benzodiazepines (BDZs), which enhance the activity of the
GABA receptor, also enhance the teratogenic effects of VA in humans [ 10 ].
Chlordiazepoxide, another BDZ, has been shown to produce NTDs in the hamster [
11 ].
Further support for the contention that VA may produce NTDs by way of GABA
activity include the following evidence. One hypothesis of VAs mechanism of
action holds that it alters intracelluar pH. This may be the case as GABA can
increase intracellular proton levels by intensifying bicarbonate ion
conductance through a GABA-gated channel [ 15 ] which may act as a
"developmental handshake" and regulate neuronal differentiation [ 16 ]. The
chloride channel, an integral part of the GABA a receptor, has been implicated
in embryonic development [ 17 ]. GABA receptors are first seen at the time the
neural tube formation [ 18 ]. Binding sites to GABA agonists and antagonists
and the expression of GABA receptor mRNAs are seen starting at 4 days of
development and peak at 10â15 days, corresponding to the time of neural tube
formation [ 19 ]. Many neurotransmitters, including GABA, are growth factor
candidates for the CNS [ 20 - 22 ].
Based on the above information we have undertaken this study to systematically
examine the effects of GABA agonists and antagonists at both the GABA a and b
receptor and the role they may play in SB. We used an established model of SB
in the rat to test the hypothesis using VA as a known standard. This model of
SB uses the width of the vertebral arch as an indicator of neural tube closure.
While this model does not produce a specific SB lesion the widening of the
vertebral arch provides a model that resembles human SB in terms of
accompanying defects [ 12 - 14 , 23 ] and response to folate and other drugs [
6 , 11 , 24 , 25 ] and meets the formal definition of a neural tube defect: any
defect in the covering of the central nervous system.
We report here the effects of GABA a and b receptor agonists and antagonists
administered to rats at 10 days gestation, the period of neural tube formation.
Results and Discussion
A total of 1156 embryos from 123 litters were examined. Measurements from the
embryos were averaged for each litter and the litter was used as the unit of
analysis. All measurements were made in a blind manner. ANOVA revealed a
significant effect of drug treatment of the average vertebral arch distance
(F(13, 109)=7.70, p < .0001). The Bonferroni test was used for follow-up
comparisons and the comparisons to the normal saline group are given below and
in Table 1 and in Figure 1 where the results are presented graphically.
Occasionally other defects were noted in the embryos, chiefly fused ribs, these
defects were not appreciable and did not impact the vertebral arch analysis.
There is no statistically significant relationship between mean vertebral arch
distance and mean littler size.
Effect of GABA receptor ligands on vertebral arch distance. Mean vertebral arch
distance for drug groups. Error bars represent standard deviations. Exact
probability differences from normal saline are given above error bars. See text
for comments.
Valproic Acid produced a significant widening of the vertebral arch in a manner
consistent with previous reports (p < .0005) [ 6 , 24 ]
The GABAa agonist muscimol produced a significant widening of the vertebral
arch at all three doses tested (1, 2, 4 mg/kg) (p=.06 for 1 mg/kg, p < .05 for
2 & 4 mg/kg). The GABAa antagonist bicuculline also widened vertebral arch
distance at the 0.5 and 1 mg/kg doses (p < .05), but not at the 2 mg/kg dose.
The GABAb agonist baclofen produced significant widening of the vertebral arch
at 30 mg/kg (p < .05) but not at 15 or 60 mg/kg. The GABAb antagonist
hydroxysaclofen produced a significant narrowing of the vertebral arch at all
three doses tested (p=.09 for 1 mg/kg, p=.001 for 3 mg/kg and p=.05 for 5
mg/kg).
This study indicates that substances active at either the GABA a or b receptors
have teratogenic potential. The most striking feature of the drug effects is
the differential effect of antagonizing the GABAb receptor with
hydroxysaclofen. While the GABAa agonist muscimol and antagonist bicucullin
widened the vertebral arch, as did the GABAb agonist baclofen, the GABAb
antagonist hydroxysaclofen narrowed the vertebral arch. Narrowing of the
vertebral arch was unexpected but has been previously reported. Previous work
in this laboratory has demonstrated narrowing of the vertebral arch when zinc
is co-administered with baclofen or muscimol [ 13 ]. It is unclear what the
structural or functional consequence of vertebral arch narrowing is. Work in
this laboratory has shown lags in neuromuscular development associated with
excessive zinc exposure during neural tube formation and, possibly,
accompanying narrowing of the vertebral arches [ 26 ]. Another curious finding
of this study is that widening of the vertebral arch occurs with either the
GABAa agonist muscimol or the antagonist bicuculline. In classic pharmacology
it would be expected that the effects would be opposite. However, in this
instance it may be that any disruption of the normal function of the GABAa
receptor and its integral chloride channel result in widening of the vertebral
arch. On the other hand the GABAb receptor is not directly linked to a chloride
channel exerting its effect via second messenger systems. This functional
difference may allow for a classic agonist-antagonist drug effect during neural
tube formation.
One potential confound of this study is the role of developmental delays.
Animals exposed to valproate and presumably suffering from widened vertebral
arches can show weight differences well into post-natal life. However,
behavioral differences persist even when weight differences no longer exist [ 1
]. However, we are unaware of any studies directly examining the spinal columns
of animals well into post-natal life, therefore it is unclear if the widening
of the vertebral arch seen at 21 days persists. Another limitation to this
study is the small sample sizes for some groups and the administration of drugs
to only one day. These results need to be replicated using larger sample sizes
and administering the drugs at other times during gestation. It is possible
that these drugs may disrupt neural tube formation if given outside of the
classic time frame for neural tube formation.
GABA is a well-documented neurotrophic agent involved in brain development [ 27
- 30 ]. Most of the work done on the effects of GABA and neural development has
been done on embryos and embryonic tissue well past the neural tube stage [ 27
, 31 , 32 ]. However, there is evidence of glutamic acid decarboxylase (GAD)
and GABA receptor expression about the time of neural tube formation [ 27 ].
Given that GABA is important to neural development, and the early developmental
time frame of the GABA system, it is logical that agents active at the GABA
receptor (ethanol, BDZs) can have adverse consequences on CNS development.
Nearly all of the substances examined are GABAa receptor ligands. GABAb
receptor effects have been little studied and the role of the GABAb receptor in
neural development is little known and should be more thoroughly investigated.
Conclusions
GABA may play an important role in neural tube formation and the production of
neural tube defects. Substances active at the GABA a or b receptor may be
potentially teratogenic. In particular, the findings indicate that GABA a or b
ligands are capable of altering neural formation. GABA may play a greater than
appreciated role in neural tube formation and may be important in neural tube
defects. The narrowing of the vertebral arch produced by the GABA b antagonist
hydroxysalcofen suggests that GABA b receptor may play an important, but
undefined role in neural tube closure that differs from the GABA a receptor.
Materials and Methods
Female Long-Evans rats 120 days of age were housed with ad-lib access to food
and rat chow (Purina, Brentwood, MO) under 12:12 light:dark conditions. The
females were mated with males of the same age and strain overnight with the
observation of a copulatory plug as evidence of mating and counted as day 0 of
pregnancy. The females were then separated and housed singly. At 10 days of
gestation the females were treated with one drug as described below. Ten days
of gestation corresponds with neural tube formation in the rat. All drugs and
chemicals were obtained from Sigma Chemical, St, Louis, MO. Doses were
empirically determined with pilot studies. This study was reviewed and approved
by the Institutional Animal Care and Use Committee of the University of
Nebraska at Kearney.
Reference groups
Normal saline intraperitoneally (IP) 0.9 cc to establish baseline.
Valproic acid subcutaneously (SC) 1200 mg/kg (600 mg/cc) in two divided doses
over 8 hours. Previous studies have shown this dose and route to reliably
produce SB in the rat [ 24 ]. This group provided a positive control to which
the teratogenic activity of other substances could be compared.
GABAa test groups
Muscimol IP at 1, 2, or 4 mg/kg (1 mg/cc). Muscimol is a well-documented
specific and potent GABAa agonist [ 33 ].
Bicuculline methiodide IP at 0.5, 1 or 2 mg/kg (1 mg/cc). Bicuculline is a
well-documented specific and potent GABAa antagonist [ 33 ].
GABAb test groups
Baclofen IP at 15, 30, or 60 mg/kg (9 mg/cc as aqueous suspension). Baclofen is
a well-documented and specific GABAb agonist [ 34 ].
Hydroxysaclofen IP at 1, 3, or 5 mg/kg (1 mg/cc). Hydroxysaclofen is a
well-documented specific and potent GABAb antagonist [ 34 ].
Embryo studies
After injection gestation was allowed to otherwise progress as normal. At 21
days gestation the females were euthenized with chloroform, the abdomen opened
and the uterus and uterine contents removed. Fetuses were removed and had their
abdomens opened to allow for the penetration of 10% 0.1 M phosphate buffered
formalin in which they were immersed. After three days fixation the fetuses
were stained for bone and cartilage as described previously [ 24 ]. Briefly,
the fetuses were eviscerated and skinned. Cartilage was stained with alcian
blue followed by bone staining with alizarin red. The fetuses were cleared in
KOH and graded concentrations of glycerol.
After clearing the fetuses were inspected with a dissecting microscope and the
width of the vertebral arch gap was measured with an eyepiece micrometer from
T-9 to S-4. Previous work has shown that these vertebrae are the most clearly
visible and the most frequently effected [ 6 , 24 ]. For the sake of clarity
vertebral arch distances from T-9 to S-4 were averaged for the each embryo.
For analysis vertebral arch distance was averaged for each fetus and then for
the litter. Data was analyzed with Analysis of Variance with follow-up
statistics (Bonferroni test) using Statview 5.0 for the Macintosh.
