INTRODUCTION Sensory deprivation has been a productive approach to investigate the... environmental stimuli on the developing brain. Lack of excitatory...

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INTRODUCTION
Sensory deprivation has been a productive approach to investigate the effects of
environmental stimuli on the developing brain. Lack of excitatory inputs may leave unaffected
some neurotransmitter systems. However, the GABAergic system seems to be regulated by sensory
input, thus revealing a very important role during the development of somatosensory pathways. For
instance, the cortex of monkeys, cats, and rats show particular changes in their GABAergic
components after different types of sensory or visual deprivation. In the rat somatosensory cortex,
deprivation from whisker input, using different paradigms, results in changes in GABAergic
circuitry elements, such as the numerical density of terminals (Micheva and Beaulieu, ’95), of
GAD-containing neurons (Akhtar and Land, ’91), and of muscimol binding to GABAA receptors
(Fuchs and Salazar, ’98). It is not known, however, if the cortical GABAB receptor population is
similarly affected by whisker trimming. The main purpose of this research is to investigate the
effects of sensory deprivation on GABAB receptor binding in the rat barrel cortex .
The rodent somatosensory whisker to barrel pathway has several features that make it a
desirable system to study sensory deprivation. For instance, there is a 1:1 topographic relationship
between each of the whiskers in the rat’s face and a group of neurons that constitute a ‘barrel’ in
layer VI of SI (Woolsey and Van der Loos, ’70): each barrel responds primarily to one principal
whisker. This feature has enabled the discovery of cytoarchitectonic (Woolsey and Van der Loos,
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’70; Welker and Woolsey, ’74; Van der Loos and Woolsey, ’73) and physiological effects (Welker,
’71, ’76; Simons, ’78; Simons and Woolsey, ’79) of whisker stimulation and/or deprivation.
Furthermore, at birth a rodent’s brain is very immature. This allows to closely follow
developmental events, such as transience of synapses (Micheva and Beaulieu, ’96),
neurotransmitters (Micheva and Beaulieu, ’95), neurotransmitter receptors (Fuchs, ) and their
subunits (Penschuck, et al., ’99) during the first postnatal weeks, and thus helps explain the
importance of timing in the appropriate formation of sensory systems. And last, surgical procedures
on the somatosensory (SI) cortex of rats and mice are relatively easy to perform, and allow for a
variety of chemical, physiological and mechanical preparations and manipulations.
Effects of sensory deprivation on GABAergic cortical circuitry have been widely studied.
Pioneer studies on the adult monkey’s visual system showed that depriving visual input from one
eye resulted in decreases of both GABA and its synthesizing enzyme GAD on the deprived cortical
neurons (Hendry and Jones, ‘86). In the SI cortex of adult rodents, similar effects of deprivation
have been observed. First of all, GAD is reduced in deprived barrels after trimming whiskers in the
adult, but not in the neonatal rat. (Akhtar and Land, ’91). Physiological studies showed that adult
rats with neonatally deprived barrel neurons show signs of disinhibition, such as higher
spontaneous activity, and a decreased selectivity to respond to a specific angle of whisker deflection
(Simons and Land, ‘87). These physiological changes remained even after allowing neonatally
deprived rats to regrow their whiskers for several weeks, indicating the dramatic, long lasting effect
of neonatal deprivation. What is the chemical basis of these physiological changes? Since GABA
is the main inhibitory neurotransmitter in cortex, it was important to consider it and its receptors as
suitable candidates responsible for these physiological changes. Blocking GABAA receptors with
the antagonist bicuculline results in signs of cortical disinhibition (Kyriazi et al, ‘96). Furthermore,
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binding of the GABA agonist muscimol, which selectively binds to GABAA receptors, is reduced in
the deprived barrels. This effect was observed in both neonatally and adult deprived rats, and was
still present even after allowing the rats to grow their whiskers for ten additional weeks after the
trimming period. Thus, these overall decreases after deprivation were suggested as a downregulating mechanism that compensates for the reduced sensory input (Fuchs and Salazar, ’98).
The contributions of GABAB receptors to the barrel circuitry have been recently studied
(Micheva and Beaulieu, ’97). Whereas GABAA receptor activation directly increases membrane
chloride conductance and allows it to move down its concentration gradient, thus hyperpolarizing
the mature postsynaptic cell, GABA inhibitory action is different through other receptors. Through
GABAB receptors, binding of GABA activates G-proteins that increase potassium and calcium
channels’ permeability, so activation of these receptors results in a slow, long-lasting
hyperpolarization of the cell’s membrane, and thus, inhibition of the postsynaptic cell. GABAB
receptors are also located presynaptically (Deisz and Prince, ’89; Deisz, ’99; Howe et al, ’87). This
ensures not only inhibition of the postsynaptic cells, but also of presynaptic neurotransmitter
release. Finally, regardless of the difficulty that variables such as affinity, competition, and nonspecific binding impose to studies of receptor distribution, different methods have been designed to
achieve such objectives. As a result, GABAB receptors in cerebral cortex have been found in all
layers of cerebral cortex, with a distribution somehow resembling that of the GABAA receptor
population (Chu et al., ’90; Bowery et al., ’87).
How does deprivation affect GABAB receptors in SI? So far, there is no evidence indicating
that these receptors change as a result of deprivation. However, two main findings allow the
formulation of a hypothesis predicting a decrease in GABAB receptors after deprivation. First, as
many as two thirds of GABA terminals in layer IV of SI cortex are lost after neonatal whisker
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trimming (Micheva and Beaulieu, ’95); thus it can be predicted that presynaptic GABAB contained
in those terminals might also be lost along with the terminals. Secondly, postsynaptic GABA
receptors, such as GABAA, decrease in numbers after sensory deprivation as a mechanism
compensating for the loss of input (Fuchs and Salazar, ’98); thus, postsynaptic GABAB receptors
could also decrease as a similar compensating mechanism.
OBJECTIVES:
The objectives of this research are to answer the following questions:
1) Are there any deprivation effects on the GABAB receptor population of barrel cortex? If
deprivation causes loss of GABAergic terminals, it can be possible to expect that some of those
terminals could contain presynaptic GABAB receptors, in which case would be also lost. In
other words, deprivation could result in loss of presynaptic GABAB receptors.
a. Does whisker trimming for six weeks result in changes in GABAB receptor binding in
barrel cortex? This same period of deprivation causes decreases in the GABAA receptor
population. There is no evidence suggesting that GABAB receptor population could be
affected differently.
b. Does trigeminal nerve transection in 2 weeks old rats result in GABAB receptor binding in
barrel cortex?
2) If there are deprivation effects, are these greater when whisker trimming is performed during
the first 6 neonatal weeks? In other words, is there a critical period involved? Normally,
transient increases in the GABAA and GABAB receptor populations appear within the first 2
postnatal weeks. Observing major decreases in these receptors after deprivation during these
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postnatal periods could uncover the importance of environmental vs intrinsic factors leading
these raises. GABAA receptors decrease similarly whether deprivation for 6 weeks is performed
starting at birth or in the adult. Could the GABAB receptor population respond any differently
to neonatal deprivation, thus uncovering some critical periods?
3) If there are deprivation effects of trimming whiskers for 6 weeks, would these effects be
reversible or would they remain after ten more weeks allowing whiskers to regrow and to be
normally used? GABAA receptor decrease after deprivation remains after 10 weeks of recovery
and GABAB receptors could respond in a similar, long lasting fashion to deprivation.
MATERIALS AND METHODS
Subjects
Subjects will be Long-Evans hooded rats (Simonsen, Gilroy, CA), distributed in four deprivation
groups. In the first group (n=6), whiskers will be trimmed from birth to postnatal day 10 (P0-P10)
to test whether normal sensory input contributes or not to the normal GABAB receptor binding
levels. Normal GABAB receptor will be assessed using either the undeprived side of the same
subjects or non deprived subjects. In the second (n=6), whiskers will be kept trimmed for six
weeks, starting at birth (P0-P41). In the third group (n=6), whiskers will be also clipped for 6 wk,
from wk 6-12 (P42-P83); this and the second group will be compared to test for a critical period. In
the fourth group (n= 6) whiskers will be kept trimmed for the first 6 postnatal weeks, followed by
10 wk without clipping, to test for reversibility of the effect, if any.
Trigeminal nerve cut (…)
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Histology
Unperfused rats will be sacrificed by decapitation. The deprived barrel region will be dissected out
from the brain, flattened at –30oC with the heat dissipator of a cryostat (28090 Frigocut N.
Reichert-Jung), and stored at -80 oC. Sections 16-20 m-thick will be cut at –20oC tangentially to
the pial surface, and will be thaw-mounted onto gelatin subbed slides. They will be air-dried for 1/2
to 3 h and then stored desiccated at –80oC. Following the ligand binding and autoradiography the
sections will be stained for cytochrome oxidase (Wong-Riley, ’79).
GABAB receptor binding
GABAB receptors will be assessed with 0.5 nM [3H]-CGP 62349 (85 Ci/mmol, American
Radiolabeled Chemicals, Inc. St. Louis, MO., U.S.A.). Methods will be based on those described
previously (Ambardekar et al., ’99). Sections stored at –80oC will be thawed and dried. In order to
remove endogenous GABA, sections will be incubated for 80 min at room temperature in 70 mM
Tris-HCl buffer (pH 7.4) containing 2.5 mM CaCl2. Sections will be then air dried for 20-30 min at
room temperature. Then they will be incubated for 60 min at room temperature in the ligand
solution, consisting of 0.5 nM [3H]-CGP 62349, 50 mM Tris HCl, and 2.5 mM CaCl2 (pH 7.4).
Non-specific binding will be assessed with addition of unlabeled antagonist CGP54626 (10 M) to
adjacent sections. Sections will then be rapidly aspirated to remove any excess binding solution,
rinsed in fresh buffer followed by a rapid rinse in distilled water, and finally air-dried.
Autoradiography
The brain sections and tritium standards (Microscale, Amersham) will be exposed simultaneously
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in the same cassette to tritium-sensitive Hyperfilm (Amersham). Following a 2-3 week exposure
period, the film will be developed with Kodak D-19 and processed according to the manufacturer’s
instructions.
Data analysis
[3H]-CGP 62349 will be quantitatively analyzed using a video-based computerized image analysis
system (MCID, Imaging Research, St. Catherine, Ont., Canada). Tritium standards will be used to
calibrate autoradiographic densities. Samples will be taken within a computer-generated circle
centered over each barrel. For each section, the size of the circle will be calculated as the mean of
the non-deprived barrels divided by the mean of the deprived barrels. A ratio for each subject will
be then calculated by averaging the ratios for each section. The group average will be converted
into percent decrease in the deprived rows. Analysis of data will include t-test and analysis of
variance, using 0.05 as the significance level.
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