CHAPTER 1
INTRODUCTION
Sensory deprivation has been a productive approach to investigate the effects of
environmental stimuli on adult and developing brain. Whereas lack of normal excitatory inputs
leaves some cortical neurotransmitter systems unaffected (Goodman et al., 1993; Schlaggar et al,
1993), it can lead to down regulation of the gamma-aminobutyric acid (GABA)-ergic system. For
instance, the cortex of monkeys, cats, and rats shows particular changes in its GABAergic
components after different types of sensory or visual deprivation. In the rat somatosensory cortex,
deprivation of whisker input results in decreases in GABAergic circuitry elements, such as the
number and proportion of GABA-immunoreactive synaptic contacts in layer IV (Micheva and
Beaulieu, 1995), of glutamic acid decarboxylase (GAD) levels (Akhtar and Land, 1991), and of
muscimol binding to GABAA receptors (Fuchs and Salazar, 1998).
Alterations in specific GABA receptors subunits might help understand GABAergic
function. This is the first investigation where whisker deprivation regulates specific GABAA
receptor subunits.
The Whisker to Barrel System of the Rat
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
1
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, 1970): each barrel responds primarily to one principal
whisker. This feature enabled the discovery of cytoarchitectonic (Woolsey and Van der Loos,
1970; Welker and Woolsey, 1974; Van der Loos and Woolsey, 1973) and physiological (Welker,
1971, 1976; Simons, 1978; Simons and Woolsey, 1979) effects of whisker stimulation and/or
deprivation, and to investigate the experience-dependent maintenance of synapses (Micheva and
Beaulieu, 1996), neurotransmitters (Micheva and Beaulieu, 1995), and neurotransmitter receptors
(Fuchs, 1995). Moreover, the somatosensory (SI) cortex of rats is accessible to surgical procedures,
allowing for a variety of chemical, physiological and mechanical preparations and manipulations,
such as measurements of changes of GABA receptor subunits after topical cortical blockade of
NMDA receptors (Penschuck, et al., 1999).
Sensory Deprivation and GABA A Receptors
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 results in decreases of both GABA and its synthesizing enzyme GAD in the deprived cortical
neurons (Hendry and Jones, 1986). 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 for 6
weeks beginning in the adult, but not beginning in the neonate (Akhtar and Land, 1991).
Physiological studies showed that simply trimming rat whiskers leads to signs of disinhibition in
the deprived barrel neurons, such as higher spontaneous activity, and a decreased selectivity to
respond to specific angles of whisker deflection (Simons and Land, 1987). These physiological
changes remain even after allowing neonatally deprived rats to regrow their whiskers for several
2
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 GABA 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, 1996) that are similar to those from deprived barrel
cortex. Furthermore, binding of the GABA agonist muscimol, which selectively binds to GABAA
receptors, is reduced in the deprived barrels (Fuchs and Salazar, 1998). 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. These overall decreases after
deprivation were suggested as a down-regulating mechanism that compensates for the reduced
sensory input (Fuchs and Salazar, 1998). Recent studies showed that whisker trimming reduced the
numerical density of both intracortical and thalamocortical symmetrical synapses, associated with
inhibitory neurons in barrel neuropil (Sadaka et al., 2003).
GABA inhibitory effects in cortex depend on the types of GABA receptors involved.
Inhibition through the GABAA receptor, a chloride channel itself, is relatively fast. These receptors,
when activated, increase the chloride conductance of the membrane, affecting transmitter release in
the presynaptic neuron, and producing hyperpolarization or shunting inhibition of the postsynaptic
neuron (MacDonald and Olsen, 1994; Xi and Akasu, 1996). Through the metabotropic GABAB
receptor inhibition is slower, and involves changes in potassium and calcium permeability at
presynaptic and postsynaptic synapses (Misgeld et al., 1995; Howe et al., 1987; Deisz and Prince,
1989; Deisz et al., 1997).
3
GABA Receptor Subunits and Sensory Deprivation
GABAA receptors subunits comprise a family of at least 17 subunits (Davies et al., 1997).
Each subunit is expressed in a particular laminar pattern in somatosensory (SI) and visual cortex
(V1). For instance, the α1 subunit, which represents the majority of the GABAA receptors, is
denser in layers III-IV (Fritschy, et al, 1994) of SI and V1. This area-specific expression might
represent distinct functional areas, which can be differentially affected by sensory deprivation
paradigms. In focal cortical malformations induced by neonatal freeze lesions, α1 and α5-GABAA
subunits decrease in rat SI (Redecker, 2000). Electrolytic lesion of thalamus in the newborn
decreases α1 in layers III-IV, but increases α5 in the same areas (Paysan, 97). Decreases in α1 have
also been observed in layer IVC of area 17 after monocular deprivation in monkeys (Huntsman
1994, Jones, 1995 and 1998), and hippocampus (Simburger et al, 2001, Chen et al, ’99, Sperk et al,
1998).
When whiskers are trimmed during early postnatal development, stimulation of the
regrown whiskers causes responses from layer II/III neurons to be reduced in the corresponding
deprived column, whereas neighboring barrels columns show stronger responses (Lendvai et al.,
2000; Stern et al., 2001). Similarly, plucking whiskers from birth results in weaker responses of
neurons in layers II/III and IV of the related barrel column (Fox, 1992, 1994), but causes stronger
responses from neighboring barrel columns (Simons and Land, 1987; Fox, 1992, 1994). In like
manner, responses from layer II/III barrel neurons are recorded in rats deprived as adults
(Glazewski and Fox, 1996; Wallace and Fox, 1999). However, the chemical basis for these
changes has yet to be determined.
Since GABAA receptors decreased in layer IV of deprived barrel neurons (Fuchs and
Salazar, 1998), these effects might not only be restricted to this layer, but to layers II and III of the
4
same deprived column. Moreover, changes in GABAA receptors, as assessed by autoradiography,
might also be paralleled by changes in GABAA receptor subunits. A favorable candidate to observe
changes might after deprivation is the α1-GABAA subunit, since it is the one that predominates in
layers II/III of barrel columns. To determine whether sensory deprivation affects GABAA
receptors on layers II/III and IV, quantitative autoradiographic tests were performed on adult
whisker trimmed rats. To determine whether the same deprivation paradigm affects the α1-GABAA
receptor subunits, immunological methods were used.
CHAPTER II
MATERIALS AND METHODS
Subjects
The subjects were 16 Long-Evans hooded rats (Simonsen, Gilroy, CA), maintained on a
12:12-hour light:dark cycle, with food and water available ad libitum. Whiskers from either the
middle row C or the other rows ABDE were trimmed for six weeks.
5
Whisker Deprivation
Six-week old rats had whiskers trimmed every other day for 6 weeks, ensuring that their
vibrissae was kept shorter than 1 mm. All experimental rats had the mystacial vibrissae in either
row C, rows ABDE, or all rows clipped on one or both sides of the face. During the procedure,
rats were unanesthetized and were hand-held with gentle restraint. These protocols were
approved by the Institutional Animal Care and Use Committee at the University of North Texas.
Histology
Unperfused rats were killed at the end of their deprivation schedules by decapitation. For
tangential sections the deprived barrel region was dissected out from the brain, and was flattened
and frozen at -44˚C with the spring loaded heat dissipater of a cryostat (2800 Frigocut N,
Reichert-Jung, Cambridge Instruments, Deerfield, IL). Cryostat sections 16 µm thick were cut at
-20˚ C tangentially to the pial surface, and were thaw-mounted onto gelatin subbed microscope
slides. For coronal sections the whole brain was frozen in -40˚C isopentane for 5 min, transferred
to -80˚C isopentane, and cut and mounted as above. Sections were air dried for 0.5 to 3 h and
then stored desiccated at -80˚C until their use for either immunochemistry or receptor
autoradiography. After receptor autoradiography, some sections were Nissl stained, to examine
whether cellular and immunological observations can be paralleled. Other sections were stained
for cytochrome oxidase (CYO) activity (Wong-Riley, 1979) to localize barrels and to analyze
any similarities between CYO activity and receptor binding.
Immunocytochemistry
6
Subunit-specific antisera were used to visualize GABA subunits. The α1-GABAA receptor
subunit antisera were raised in rabbit against synthetic peptides derived from mouse subunit cDNA
(generously provided by J.M. Fritschy).
Prior to the immunological procedure, slides with rat sections were transferred to a 10˚C
refrigerator for 5-10 min. Then they were dried in a slide warmer for 45 sec, immersed horizontally
in 0.5% paraformaldehyde in phosphate buffer (0.15M) pH 7.4 solution (Fristchy and Mohler,
1995) for 20 min, and rinsed in ice-cold 0.5M Tris-saline buffer (TBS), pH 7.6 for 10 min. This
and all of the following steps were performed with gentle agitation using a rocker table. Slides
were then washed in preincubation solution consisting of TBS containing 10% normal goat serum
and 0.05% Triton X-100.
Sections were and incubated for two days and two nights at 4˚C in primary antibody
solution (1:100,000) diluted in TBS buffer containing 10% normal goat serum (NGS). Sections
were then washed three times for 1.5 hr in TBS and incubated for 30 min at room temperature in
TBS containing 2% NGS and goat-raised biotinylated secondary antibodies (
)diluted 1:300 in
TBS pH 7.4. After 1 hr additional washing in TBS, sections were transferred to the avidinperoxidase solution (Vectastatin Elite kit; Vector Laboratories, Burlingame, CA) for 30 min,
washed and processed using diaminobenzidine hydrochloride (DAB) as chromogen. Sections were
air-dried, dehydrated with ascending series of ethanol, cleared in xylenes and coverslipped using
DPX.
Ligand binding
GABAA receptors were assessed using [3H]muscimol (Mower et al., 1986; Schwark et al.,
1994). This sections had been previously used to determine changes in layer IV after deprivation
(Fuchs and Salazar, 1998). Sections were preincubated 20 minutes at 4oC in 0.31 M Tris-citrate
7
buffer, pH 7.1, and then were incubated for 40 minutes at 4oC in the buffer containing 10 nM
[3H]muscimol (12-20 Ci/mmol; DuPont NEN, Boston, MA). Sections then were rinsed twice for
30 seconds in cold buffer, dipped briefly in dH20, and dried in a stream of air. Nonspecific binding
was assessed in the presence of 1 mM GABA.
Autoradiography
The brain sections and tritium standards (Microscale, Amersham, Arlington Heights, IL)
were exposed simultaneously in the same cassette to tritium-sensitive Hyperfilm-3H (Amersham).
Following a 2-4 month exposure period, the film was developed with Kodak D-19 and processed
further according to the manufacturer’s instructions.
Data analysis: Immunostained and Nissl stained sections
For semiquantitative image analysis, sections were digitized using a video-based
computerized image analysis system (MCID, Imaging Research, St. Catherine, Ont., Canada).
Measures of relative optical density were obtained from all the tangential α1-GABAAimmunostained sections, from cortical layer I (whenever possible) to layer IV. Samples were taken
within a computer-generated circle over each barrel column, allowing for comparisons between
deprived and intact rows.
Within each section, the mean ratio of densities in deprived:nondeprived rows was
determined. The average ratio for each subject was then calculated from the section means. Then
the average ratio for all subjects was obtained. To test the null hypothesis that this ratio was not
8
different from 1, Student’s t-test (two tailed)was used. Experimental groups were compared with
one another by using analyses of variance with post hoc t-test corrections. The significance level
was 0.05. Percentage of decrease within each section was calculated as the mean for for deprived
barrels minus the mean for nondeprived barrels, divided by the mean for nondeprived barrels. For
display, images were contrast-enhanced in pseudocolor.
Data analysis: Autoradiographs
[3H]Muscimol was quantitatively analyzed using a video-based computerized image
analysis system (MCID M-4, Imaging Research, St. Catharines, Ont., Canada). Tritium standards
were used to calibrate autoradiographic densities. Ligand binding densities were automatically
calibrated by using a best-fit equation based on the plastic tritium standards, which had been
calibrated in nCi/mg tissue wet weight based on standards made from rat brain mash (Fuchs and
Schwark, 1993). The analysis of data followed the same steps as the immunostained and Nissl
stained sections.
CHAPTER III
RESULTS
Effects of Whisker Deprivation on α1-GABAA receptor subunit
Deprived barrel columns showed overall less immunoreactivity for α1-GABAA receptor
subunit than adjacent intact barrel columns. This difference could be readily detected even in the
most superficial cortical layers II/III corresponding to a deprived cortical column, and remained in
layer IV. Control subjects showed no difference between intact C and adjacent rows.
9
Nissl Stainning
Deprivation also resulted in lower staining density in deprived columns. Nissl staining
reduction was apparent in superficial layers II/III from deprived columns. Deprived layer IV also
showed less staining than adjacent intact rows in layer 4, but the difference could not be seen with
the unaided eye as easily.
GABAA receptor autoradiography
Muscimol binding showed an overall reduction in the deprived columns as compared to the
adjacent intact ones. This difference was present in cortical layers II/III and IV.
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Department of Physiology, Kurume University School of Medicine, Japan
16