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
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the antagonist bicuculline results in signs of cortical disinhibition (Kyriazi et al, ‘96). Furthermore,
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
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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
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:
1) To evaluate the effects of whisker trimming of rats from birth to 6 weeks of age on the GABAB
receptor binding of the corresponding cortical barrels.
2) To evaluate the effects of transecting the adult rat’s infraorbital branch from the left trigeminal
nerve on the GABAB receptor binding in the deprived cortical layers II-VI, after one and two
post-operative weeks.
3) To obtain the time courses of GABA A and GABAB receptor binding in barrel cortex of adult
rats, with C-whiskers trimmed for 1, 2, 3 and 5 weeks. This will aid in determining the smallest
period sufficient to elicit the largest deprivation effect.
MATERIALS AND METHODS
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Subjects
Subjects will be Long-Evans hooded rats (Simonsen, Gilroy, CA. For completion of the first
objective (n=6), whiskers from C-row will be trimmed from birth to postnatal week 6 (P0-PW6) to
test whether normal sensory input might contribute to normal GABAB receptor binding. Normal
levels of GABAB receptor binding will be assessed using the undeprived adjacent rows B and D.
For the second objective (n=6) the infraorbital branch of the trigeminal nerve will be transected on
the left side of adult rats. These rats will be allowed to live for one or two more weeks after surgery.
For the time course study adult rats will have their whiskers trimmed for one, two, three, and five
weeks (n=2, 2, 4 and 4, respectively).
Histology
Unperfused rats will be sacrificed by decapitation. In C-row deprived animals 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. In infraorbital nerve transection
animals, the brain will be dissected out and immediately frozen in –30oC isopentane. Coronal
sections 16-20 m-thick will be cut at –20oC using the same cryostat. Following the ligand
binding and autoradiography the sections will be stained for cytochrome oxidase (Wong-Riley,
’79).
GABAB receptor binding
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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.) or CGP 54626. 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
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
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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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