Γ-Aminobutyric acid in adult brain: an update.

This review assesses the parallel literature on the role of gamma - aminobutyric acid (GABA) in brain plasticity and GABA elements dysfunction related disorders. I review historical and new data from both animal and human sources which have helped define the key role for this transmitter synthesis, release and reuptake, GABA receptors subtype regulation, and GABAergic neurons function in the adult brain. The role of GABAergic elements in neurological and psychiatric disorders is briefly discussed.

CB1 Cannabinoid Receptor Expression in the Barrel Field Region Is Associated with Mouse Learning.

We found previously that fear conditioning by combined stimulation of a row B facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) leads to expansion of the cortical representation of the "trained" row, labeled with 2-deoxyglucose (2DG), in the layer IIIb/IV of the adult mouse the primary somatosensory cortex (S1) 24 h later. We have observed that these learning-dependent plastic changes are manifested by increased expression of somatostatin, cholecystokinin (SST+, CCK+) but not parvalbumin (PV+) immunopositive interneurons We have expanded this research and quantified a numerical value of CB1-expressing and PV-expressing GABAergic axon terminals (CB1+ and PV+ immunopositive puncta) that innervate different segments of postsynaptic cells in the barrel hollows of S1 cortex. We used 3D microscopy to identify the CB+ and PV+ puncta in the barrel cortex "trained" and the control hemispheres CS+UCS group and in controls: Pseudoconditioned, CS-only, UCS-only, and naive animals. We have identified that (i) the association between whisker-shock "trained" barrel B hollows and CB1+, but not PV+ puncta expression remained significant after Bonferroni correction, (ii) CS+UCS has had a significant increasing effect on expression of CB1+ but not PV+ puncta in barrel cortex "trained" hemisphere, and (iii) the pseudoconditioning had a significant decreasing effect on expression of CB1+, but not on PV+ puncta in barrel cortex, both trained and untrained hemispheres. It is correlated to disturbing behaviors. The results suggest that CB1+ puncta regulation is specifically linked with mechanisms leading to learning-dependent plasticity in S1 cortex.

Neurochemical correlates of functional plasticity in the mature cortex of the brain of rodents.

It is commonly accepted that increase of input to sensory structures in mammals is known to produce marked changes in cortical recipient areas. This paper reviews the data concerning manifestations of changes in primary somatosensory cortex of adult animals caused by classical conditioning with reinforcement: aversive (whisker-shock) and appetitive (whisker-water) trainings. These include: anatomical, electrophysiological responses, receptor autoradiography, expression of GABA, GAD at mRNA and protein levels, expression of neuronal and astroglial GAT-1 puncta and inhibitory synaptogenesis in the hollows of "trained" barrels of the adult mouse. Here we have quoted the discovery in an earlier work of the creation of a picture of the extended perimeter of the neuronal mechanisms of coding and mediating in experience-dependent changes in the barrel cortex.

Effect of Associative Learning on Memory Spine Formation in Mouse Barrel Cortex.

Associative fear learning, in which stimulation of whiskers is paired with mild electric shock to the tail, modifies the barrel cortex, the functional representation of sensory receptors involved in the conditioning, by inducing formation of new inhibitory synapses on single-synapse spines of the cognate barrel hollows and thus producing double-synapse spines. In the barrel cortex of conditioned, pseudoconditioned, and untreated mice, we analyzed the number and morphological features of dendritic spines at various maturation and stability levels: sER-free spines, spines containing smooth endoplasmic reticulum (sER), and spines containing spine apparatus. Using stereological analysis of serial sections examined by transmission electron microscopy, we found that the density of double-synapse spines containing spine apparatus was significantly increased in the conditioned mice. Learning also induced enhancement of the postsynaptic density area of inhibitory synapses as well as increase in the number of polyribosomes in such spines. In single-synapse spines, the effects of conditioning were less pronounced and included increase in the number of polyribosomes in sER-free spines. The results suggest that fear learning differentially affects single- and double-synapse spines in the barrel cortex: it promotes maturation and stabilization of double-synapse spines, which might possibly contribute to permanent memory formation, and upregulates protein synthesis in single-synapse spines.

Increases in the numerical density of GAT-1 positive puncta in the barrel cortex of adult mice after fear conditioning.

Three days of fear conditioning that combines tactile stimulation of a row of facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) expands the representation of "trained" vibrissae, which can be demonstrated by labeling with 2-deoxyglucose in layer IV of the barrel cortex. We have also shown that functional reorganization of the primary somatosensory cortex (S1) increases GABAergic markers in the hollows of "trained" barrels of the adult mouse. This study investigated how whisker-shock conditioning (CS+UCS) affected the expression of puncta of a high-affinity GABA plasma membrane transporter GAT-1 in the barrel cortex of mice 24 h after associative learning paradigm. We found that whisker-shock conditioning (CS+UCS) led to increase expression of neuronal and astroglial GAT-1 puncta in the "trained" row compared to controls: Pseudoconditioned, CS-only, UCS-only and Naïve animals. These findings suggest that fear conditioning specifically induces activation of systems regulating cellular levels of the inhibitory neurotransmitter GABA.

Fear learning increases the number of polyribosomes associated with excitatory and inhibitory synapses in the barrel cortex.

Associative fear learning, resulting from whisker stimulation paired with application of a mild electric shock to the tail in a classical conditioning paradigm, changes the motor behavior of mice and modifies the cortical functional representation of sensory receptors involved in the conditioning. It also induces the formation of new inhibitory synapses on double-synapse spines of the cognate barrel hollows. We studied density and distribution of polyribosomes, the putative structural markers of enhanced synaptic activation, following conditioning. By analyzing serial sections of the barrel cortex by electron microscopy and stereology, we found that the density of polyribosomes was significantly increased in dendrites of the barrel activated during conditioning. The results revealed fear learning-induced increase in the density of polyribosomes associated with both excitatory and inhibitory synapses located on dendritic spines (in both single- and double-synapse spines) and only with the inhibitory synapses located on dendritic shafts. This effect was accompanied by a significant increase in the postsynaptic density area of the excitatory synapses on single-synapse spines and of the inhibitory synapses on double-synapse spines containing polyribosomes. The present results show that associative fear learning not only induces inhibitory synaptogenesis, as demonstrated in the previous studies, but also stimulates local protein synthesis and produces modifications of the synapses that indicate their potentiation.

Impairment of experience-dependent cortical plasticity in aged mice.

This study addresses the relationship between aging and experience-dependent plasticity in the mouse somatosensory cortex. Plasticity in the cortical representation of vibrissae (whiskers) was investigated in young (3 months), mature (14 months) and old (2 years) mice using [14C]2-deoxyglucose (2-DG) autoradiography. Plastic changes were evoked using two experimental paradigms. The deprivation-based protocol included unilateral deprivation of all but one row of whiskers for a week. In the conditioning protocol the animals were subjected to classical conditioning, where tactile stimulation of one row of whiskers was paired with an aversive stimulus. Both procedures evoked functional plasticity in the young group, expressed as a widening of the functional cortical representation of the spared or conditioned row. Aging had a differential effect on these two forms of plasticity. Conditioning-related plasticity was more vulnerable to aging: the plastic change was not detectable in mature animals, even though they acquired the behavioral response. Deprivation-induced plasticity also declined with age, but some effects were persistent in the oldest animals.

Rapid, learning-induced inhibitory synaptogenesis in murine barrel field.

The structure of neurons changes during development and in response to injury or alteration in sensory experience. Changes occur in the number, shape, and dimensions of dendritic spines together with their synapses. However, precise data on these changes in response to learning are sparse. Here, we show using quantitative transmission electron microscopy that a simple form of learning involving mystacial vibrissae results in approximately 70% increase in the density of inhibitory synapses on spines of neurons located in layer IV barrels that represent the stimulated vibrissae. The spines contain one asymmetrical (excitatory) and one symmetrical (inhibitory) synapse (double-synapse spines), and their density increases threefold as a result of learning with no apparent change in the density of asymmetrical synapses. This effect seems to be specific for learning because pseudoconditioning (in which the conditioned and unconditioned stimuli are delivered at random) does not lead to the enhancement of symmetrical synapses but instead results in an upregulation of asymmetrical synapses on spines. Symmetrical synapses of cells located in barrels receiving the conditioned stimulus also show a greater concentration of GABA in their presynaptic terminals. These results indicate that the immediate effect of classical conditioning in the "conditioned" barrels is rapid, pronounced, and inhibitory.

Characterization and plasticity of the double synapse spines in the barrel cortex of the mouse.

The somatosensory barrel cortex of rodents and its afferent pathway from the facial vibrissae is a very useful model for studying neuronal plasticity. Dendritic spines are the most labile elements of synaptic circuitry and the most likely substrate of experience-dependent alterations in neuronal circuits in cerebral cortex. We characterized morphologically and numerically a specific population of spines, i.e. double synapse spines, which have two different inputs--one excitatory and the other inhibitory, in the B2 barrel of mouse somatosensory cortex. We also described changes in morphology of double synapse spines induced by classical conditioning in which stimulation of vibrissae was paired with a tail shock. The analysis was carried out by means of serial EM micrograph reconstruction. We showed that double spines account for about 10% of all analyzed spines. The morphology of a typical double synapse spine is similar to the morphology of single synapse spine and both consist of two parts--a large head and a narrow, long neck. Excitatory synapses are preferentially located on the head of double synapse spines and inhibitory synapses are usually located on the neck of these spines. The length of the double synapse spine neck decreases and the cross-section area of the spine neck increases significantly as a result of sensory conditioning.

GAD67-positive puncta: contributors to learning-dependent plasticity in the barrel cortex of adult mice.

We have previously shown that a classical aversive conditioning paradigm involving stimulation of a row of facial vibrissae (whiskers) in the mouse produced expansion of the cortical representation of the activated vibrissae ("trained row"). This was demonstrated by labeling with 2-deoxyglucose (2DG) in layer IV of the barrel cortex. We have also shown that functional reorganization of the S1 cortex is accompanied by increases in the density of small GABAergic cells, and in GAD67 mRNA in the hollows of barrels representing the "trained row". The aim of this study was to determine whether GAD67-positive puncta (boutons) are affected by learning. Unbiased optical disector counting was applied to sections from the mouse barrel cortex that had been immunostained using a polyclonal antibody against GAD67. Quantification of the numerical density of GAD67-positive boutons was performed for four groups of mice: those that had been given aversive conditioning, pseudoconditioned mice with random application of the unconditioned stimulus, mice that had received only whisker stimulation, and naive animals. This study is the first to demonstrate that learning-dependent modification of mature somatosensory cortex is associated with a 50% increase in GAD67-positive boutons in the hollows of "trained" barrels compared with those of control barrels. Sensory learning seems to mobilize the activity of the inhibitory transmission system in the cortical region where plastic changes were previously detected by 2DG labeling.

Learning-induced plasticity of cortical representations does not affect GAD65 mRNA expression and immunolabeling of cortical neuropil.

Two forms of glutamic acid decarboxylase (GAD) are present in inhibitory neurons of the mammalian brain, a 65-kDa isoform (GAD65) and a 67-kDa isoform (GAD67). We have previously found that GAD67 is upregulated during learning-dependent plasticity of cortical vibrissal representations of adult mice. After sensory conditioning involving pairing stimulation of vibrissae with a tail shock, the increase in mRNA expression and density of GAD67-immunoreactive neurons was observed in barrels representing vibrissae activated during the training. In the present study, using the same experimental model, we examined GAD65 mRNA and protein levels in the barrel cortex. For this purpose, we used in situ hybridization and immunohistochemistry. No changes in the level of GAD65 mRNA expression were detected after the training. The pattern of GAD65 mRNA expression was complementary to that observed for GAD67. Immunocytochemical analysis found no changes in immunolabeling of neuropil of the barrels representing the vibrissae activated during the training. The results show that, in contrast to GAD67, cortical plasticity induced by sensory learning does not affect the expression of GAD65.