Interneurons containing somatostatin are affected by learning-induced cortical plasticity.

The maintenance of neural circuit stability is a dynamic process that requires the plasticity of many cellular and synaptic components. By changing the excitatory/inhibitory balance, inhibitory GABAergic plasticity can regulate excitability, and contribute to neural circuit function and refinement in learning and memory. Increased inhibitory GABAergic neurotransmission has been shown in brain structures involved in the learning process. Previously, we showed that classical conditioning in which tactile stimulation of one row of vibrissae (conditioned stimulus, CS) was paired with a tail shock (unconditioned stimulus, UCS) in adult mice results in the increased density of GABAergic interneurons and increased expression of glutamic acid decarboxylase (GAD)-67 in barrels of the "trained" row cortical representation. In inhibitory neurons of the rat cortex GAD co-localizes with several proteins and peptides. We found previously that the density of the parvalbumin (GAD+/Prv+)-containing subpopulation is not changed after conditioning. In the present study, we examined GABAergic somatostatin (Som)-, calbindin (CB)- and calretinin (CR)-positive interneurons in the cortical representation of "trained" vibrissae after training. Cells showing double immunostaining for GAD/Som, GAD/CR and GAD/CB were counted in the barrels representing vibrissae activated during the training and in control, untouched rows. We found a substantial increase of GAD/Som-containing cells in the trained row representation. No changes in the density of GAD/CR or GAD/CB neurons were observed. These results suggest that Som-containing interneurons are involved in learning-induced changes in the inhibitory cortical network.

Short-term sensory learning does not alter parvalbumin neurons in the barrel cortex of adult mice: a double-labeling study.

We have previously reported that a classical conditioning paradigm involving stimulation of a row of facial vibrissae produced expansion of the cortical representation of the activated vibrissae ("trained row"), this was 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 is accompanied by an increase in the density of small GABAergic cells and glutamate decarboxylase 67-positive neurons in the hollows of barrels representing the "trained row." GABA neurons of the rat neocortex co-localize with calcium-binding proteins [parvalbumin, carletinin, calbindin D28k] and neuropeptides (vasoactive intestinal polypeptide, somatostatin). In the present study we have examined GABAergic parvalbumin-positive, interneurons in the cortical representation of "trained" facial vibrissae after short-term aversive training, in order to determine whether the observed changes in glutamate decarboxylase 67-positive neurons are accompanied by changes in parvalbumin-positive neurons. Using double immunofluorescent staining, it was found that (i) all parvalbumin-positive neurons in the barrel hollows were glutamate decarboxylase 67-positive, (ii) following aversive training density of glutamate decarboxylase 67-positive neurons in barrel hollows increased significantly compared with controls and (iii) density glutamate decarboxylase 67-positive/parvalbumin-positive neurons in "trained" barrel hollows did not change compared with controls. This study is the first to demonstrate that the density of double-labeled glutamate decarboxylase 67-positive/parvalbumin-positive neurons does not alter during cortical plasticity, thus suggesting that some other population (i.e. parvalbumin negative) of GABAergic interneurons is involved in learning-dependent changes in layer IV of the barrel cortex.

Experience-dependent changes in cortical whisker representation in the adult mouse: a 2-deoxyglucose study.

Sensory experience and learning can modify cortical body maps. We have previously reported that 3 days of classical conditioning, in which stimulation of a row of whiskers was paired with tail shock, produced an expansion of the cortical representation of the "trained row" labeled with 2-deoxyglucose (2DG), in layer IIIb and IV of the barrel cortex. The present study examined plastic remodelling of the vibrissal cortical representation after pairing whisker stimulation with a drop of sweet water. Cortical representations of rows of whiskers were mapped by 2DG autoradiography after 3 days and 2 months of training. The training resulted in enlargement of the cortical representation of vibrissae involved in the stimulus pairing compared with the contralateral representation of a row of whiskers, that were not touched during the training. This modification of whisker representation was different after short-term and long-term appetitive training. After three pairing sessions, changes in the width of cortical representation were visible in layers II/IIIa (29%) and layers V/VI (28%). After 2 months of training, significant changes in the width of cortical representation row B were found only in layer IV (41%). The changes were not observed in animals, that received whisker stimulation alone or in those who were subjected to training with unpaired stimuli. The results demonstrate that stimulus-pairing-induced changes in cortical whisker representation appeared with different time courses at different levels of cortical columnar information processing.

Rapid regulation of GAD67 mRNA and protein level in cortical neurons after sensory learning.

Cortical representations of different modalities can be modified by sensory learning. Our previous studies in the barrel cortex showed that expansion of the cortical representation of a row of vibrissae could be induced by pairing stimulation of a row of vibrissae with a tail shock. The plastic change in cortical reactivity to the input used during the training was accompanied by increased density of GABA immunoreactive neurons in the involved row of cortical barrels. Using the same paradigm, the present study examined the pathway of GABA synthesis-expression of GAD67 mRNA and immunoreactivity of GAD67 isoenzyme in the barrel cortex of mice after sensory learning. In situ hybridization revealed that the GAD67 mRNA level was elevated in one row of barrels in the trained group as well as in controls receiving vibrissae stimulation alone. In contrast, elevation of immunoreactivity of the GAD67 protein occurred only in the trained group. The density of GABA-immunoreactive neurons in the hollows of barrels representing the row of vibrissae activated during the training was increased by 50%. These data indicated that sensory stimulation alone affected expression of the 67 kDa glutamate decarboxylase isoenzyme synthesis pathway, whereas the processes involved in cortical plasticity induced by associative learning modified this pathway additionally at the level of translation.

GABA immunoreactivity in mouse barrel field after aversive and appetitive classical conditioning training involving facial vibrissae.

We have previously reported that a classical conditioning paradigm involving stimulation of a row of facial vibrissae produced an expansion of the cortical representation of the "trained row", labeled with 2-deoxyglucose (2DG), in layer IV of the barrel field. The present study has examined the pattern of GABA immunoreactivity (GABA-IR) in the cortical representation of row B of the facial vibrissae after (i) 3 days of aversive training, and (ii) 2 months of appetitive training, where stimulation of row B of vibrissae on one side of the snout was used as a conditioned stimulus. The most notable observation was a greater density of GABA-IR cells concentrated in the hollows of the "trained row" B barrels compared to the hollows in the barrel field of the opposite hemisphere in the same mouse. After aversive training, we noted a 2-fold increase in the density of GABA-IR neurons in the hollows of row B; after reward training, the increase amounted to 49%. In contrast, GABA-IR was unchanged in the control groups, which received only stimulation of vibrissae without the unconditioned stimulus. The classification of labeled neurons according to size revealed that the increase in density of GABA-IR neurons was confined to the small (12-15 microm) diameter group. We concluded that the GABAergic system undergoes up-regulation, after both associative learning paradigms, and that the population of small, GABAergic neurons plays an active role in use-dependent plasticity.

Learning-induced expansion of cortical maps--what happens to adjacent cortical representations?

The present study was designed to investigate the effects of learning-dependent enlargement of cortical representation of a row of vibrissae upon the appearance of adjacent cortical representations. We have found previously that three sessions of classical conditioning, during which stimulation of row B of vibrissae is paired with a tail shock, result in an increase of cortical representation of the trained row, as visualized with 2-deoxyglucose (2DG) metabolic functional mapping. In the present experiment, after the training in which row B was stimulated, we mapped with 2DG the cortical representations of rows A and C, not stimulated during the training. We found that these representations do not differ from normal. Because of expansion of cortical representation of the trained row, the overlap between representations of neighboring rows of vibrissae became greater and the two maps co-existed within the same cortical space.

Short-lasting classical conditioning induces reversible changes of representational maps of vibrissae in mouse SI cortex--a 2DG study.

It has been known for several years that receptive field properties of sensory cortical neurons can be altered by learning experiences. We attempted to visualize a global change of the cortical body map induced by learning. In order to do this a short-duration classical conditioning involving stimulation of a row of mystacial vibrissae in mice was followed with 2-deoxyglucose (2DG) mapping of functional activity. Three conditioning sessions that paired stimulation of a row of whiskers with a tail shock produced an increase of the functional representation in somatosensory cortex (SI) of a row of the whiskers stimulated during the training. This plastic change of vibrissal representation in SI was visualized with 2DG autoradiography a day after completion of training. The expansion of representation was the most pronounced in cortical layer IV, and to a lesser extent, in layer IIIb. The expansion was observed in conditioned but not in pseudoconditioned mice or in animals that received only the conditioned stimulus. If training was discontinued, the enlargement of vibrissal representation progressively faded. The reversal could be accelerated by a behavioral extinction procedure. This study gives the pictorial demonstration of rapid, transient, and extinguishable learning-dependent changes in SI cortical maps.

Overlap of sensory representations in rat barrel cortex after neonatal vibrissectomy.

Cortical representation of the common fur of mystacial pad is situated outside postero-medial barrel subfield (PMBSF) in rat primary somatosensory cortex. Following neonatal vibrissectomy, stimulation of the common fur activates the neurones in PMBSF. We examined if sparing of one mystacial vibrissa from the neonatal ablation, which results in a very extensive increase of its cortical representation, would prevent the invasion of the common fur inputs into the PMBSF. The cortical representations were mapped with 2-deoxyglucose (2DG). It was found that six weeks after neonatal vibrissectomy sparing C3 vibrissa and common fur inputs were represented in the PMBSF. Their representation shifted from its normal location into the barrel field. This effect was observed in cortical layers II/III, IV and V.

Partial blocking of NMDA receptors restricts plastic changes in adult mouse barrel cortex.

Changes of cortical body maps can be evoked in brains of adult animals by injury to sensory nerves. We investigated changes of functional representation of row C of mystacial vibrissae in the barrel cortex of mice. Plastic changes of cortical representations were mapped with 2-deoxyglucose autoradiography. Seven days after lesions of all vibrissae except row C, cortical representation of the spared row increased in width by 60%. Partial blocking of N-methyl-D-aspartate (NMDA) receptors by subdural implants of thin sheets of Elvax impregnated with DL-2-amino-5-phosphonovaleric acid (APV) prevented development of the increase of row C representation. Low level of NMDA receptor blocking did not affect significantly the basal level of 2DG uptake and stimulus evoked uptake but prevented the plastic change of the body map.

Temporal dynamics and regional distribution of [14C]serine uptake into mouse brain.

In order to examine the uptake of L-serine into brain structures and brain metabolic compartments, L-[U-14C]serine was injected into tail vein of mice. The uptake was examined 30 min, 90 min, 3 h and 5 h after injection by both quantitative autoradiography of coronal brain sections and by biochemical analysis. Brain radioactivity was extracted and partitioned into protein associated pellets, metabolites soluble in aqueous phase and lipids soluble in the organic phase. Most of the radioactivity was found in the aqueous phase, about 10% was incorporated into lipids. Among phospholipids the highest label was found in phosphatidylserine, then in phosphatidylethanolamine and in phosphatidylcholine, it amounted to 52%, 30% and 18% of label by 90 min after injection, respectively. The brain distribution of L-serine uptake resembled that described for strychnine-insensitive [3H]glycine binding, with cortical structures being preferentially labelled.

Plasticity of mystacial fur representation in SI cortex of adult vibrissectomized rats--a 2DG study.

Localization of cortical representation of the common fur of mystacial pads was mapped with 2-deoxyglucose autoradiography in adult rats after vibrissectomy. Normal representation of mystacial fur was found in locations outside the posteromedial barrel subfield (PMBSF). Vibrissectomy performed in young adult resulted in fur inputs activating the PMBSF. This plastic change took more than two weeks and less than one month to complete.

Reduction of GABAA receptor binding of [3H]muscimol in the barrel field of mice after peripheral denervation: transient and long-lasting effects.

The effect of peripheral sensory deprivation upon GABAA receptor binding of [3H]muscimol was investigated in the barrel cortex--cortical representation of mystacial vibrissae of mice--by means of in vitro quantitative autoradiography. Unilateral lesions of all vibrissae or selected rows of whiskers were performed neonatally or in adulthood. [3H]muscimol binding was examined after various survival times up to 60 days. Both types of lesions performed in adult mice resulted in a transient decrease (10-25%) of binding values in the deafferented areas of the barrel field as compared with the unoperated control side. Sixty days after denervation [3H]muscimol binding returned to control values. Similar results were found after neonatal removal of all vibrissae. Neonatal lesion of selected rows of vibrissae, however, resulted in a decrease of [3H]muscimol binding (by about 26%) lasting up to 60 days in corresponding rows of barrels. This last result was accompanied by severe cytoarchitectonic malformation of the barrel field. The results support the hypothesis that a decrease of inhibition plays a facilitatory role in the plastic reorganization of cortical circuitry.

Short term changes of cortical body maps following partial vibrissectomy in adult mice.

Vibrissae-to-barrels pathway is often used as a model for investigating CNS plasticity. We examined early changes in the cortical representation of row C of vibrissae in adult mice, following vibrissectomy removing all whiskers except row C. The changes of cortical representation of the spared row of vibrissae were mapped with 2-deoxyglucose autoradiography. We found that one day after lesion of vibrissal follicles the areal extent of cortical representation of row C is smaller than normally, but 7 days post lesion it increases significantly, by 60 to 90%, in all cortical layers. Additionally, seven days post-lesion the intensity of labelling was increased in cortical layer V. The result suggest that plasticity that can be observed with 2-deoxyglucose mapping in the barrel cortex is not due to unmasking of existing connection, but to reorganization of connectivity at many levels.

Loss of glutamate immunoreactivity from mouse first somatosensory (SI) cortex following neonatal vibrissal lesion.

Whisker follicles were surgically ablated (lesioned) on two entire rows (B and C) of the left snout of two groups of Swiss mice, in the first 2 days after birth (neonatally lesioned) with the animals being allowed to survive for 4 weeks. In the second group at 8 weeks of age (adults), the whisker follicles of rows B and C were similarly lesioned and a survival period of 3 days allowed. Glutamate-immunoreactivity (Glu-IR) was examined in tangential sections of the first somatosensory (SI) 'barrel' cortex of these two groups (at which time it was also confirmed that the follicles had not regrown). In the neonatally lesioned mice, barrel rows B and C were more poorly defined in the right (experimental) hemisphere sections and a semi-quantitative study showed that there was a decrease in Glu-IR cell number (up to 41%) in rows B and C of the right hemisphere compared to the spared barrel rows (A and D). The loss appears to occur over almost the entire area of each deprived barrel rather than being confined to the sides or hollows. In contrast to neonatally lesioned animals, the barrels of the adult-lesioned mice appeared intact and visually similar in both the experimental (right) and control hemispheres (left). The only notable change in Glu-IR observed in the adult-lesioned animals was a decrease of 38% in the number of Glu-IR cells in the sides between the two deafferented rows of barrels (B and C), compared to the cell number between the unaffected barrels (D and E), a change also seen in the neonatally lesioned mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic conversion of phosphatidylserine via phosphatidylethanolamine into phosphatidylcholine in rat brain.

Exogenous, liposomal [14C]phosphatidylserine, and that synthesized from [14C]serine, were very slowly metabolized in cortex and hippocampus slices of rat brain; phosphatidylethanolamine (PE) formed from phosphatidylserine (PS) was not methylated to phosphatidylcholine (PC) for up to 6 hours of incubation. Among homogenates prepared from 7 separate brain regions, the cerebellum showed the highest, and the striatum and pons the lowest rate of PS synthesis and its further decarboxylation to PE; in all of these regions the stepwise methylation of PE to PC was very low. Isolated microsomal and mitochondrial fractions of whole rat brain, mixed together and incubated with [14C]serine and S-adenosylmethionine, displayed a high level of newly synthesized mitochondrial PE, and a low level of methylated PC in the microsomes. Moreover, PE formed in brain microsomes by the base exchange reaction was converted into PC in an insignificant range. These data show a limited activity for sequential methylation of PE into PC in rat brain, and suggest that it is probably not caused by the slow movement of mitochondrial PE.

Functional plasticity and neurotransmitter receptor binding in the vibrissal barrel cortex.

A form of activity dependent, functional plasticity can be induced in the barrel cortex by sensory deprivation without damage to the sensory receptors. Changes of cortical representation of a spared C3 vibrissa, when all other whiskers were plucked out, were mapped with 2-deoxyglucose autoradiography in mice and rats after a short-lasting deprivation. An increase in the volume of cortical column activated by the spared vibrissa was found already after one week of deprivation. We have found previously that if deprivation is commenced immediately after birth, the changes in cortical representation of the spared whisker appeared in the third week of life. In search of a possible reason for the delayed expression of functional plasticity in neonatal animals we examined the developmental curves of neurotransmitter receptor binding for several transmitters though to be involved in plastic processes of the cerebral cortex. We found that the beta noradrenergic and muscarinic cholinergic receptor binding increased rapidly at the end of the second postnatal week and subsequently remained high. By contrast, the metabotropic glutamate receptor binding decreased during the first month of postnatal development. The AMPA receptors binding values rose during the first two weeks of life, and then decreased. Together with our previous data on the development of NMDA and GABAa receptor and voltage dependent calcium channel binding, the results suggest that slow development of functional plasticity in neonatal animals may be due to low levels of receptors of several neurotransmitters implicated in brain plasticity.

Loss of gamma-aminobutyric acid (GABA) immunoreactivity from mouse first somatosensory (SI) cortex following neonatal, but not adult, denervation.

Whisker follicles were surgically ablated (lesioned) on two entire rows (B and C) of the left snout of two groups of Swiss mice, in the first 2 days after birth, and in the second group at 8 weeks of age (adults). Two months after surgery GABA-immunoreactivity (GABA-IR) was examined in tangential sections of the first somatosensory (SI) 'barrel' cortex of these two groups (at which time it was also confirmed that the follicles had not regrown). In the adult-lesioned mice all of the barrels appeared intact and visually similar in both the experimental (right) and control (left) hemispheres. The staining pattern of immunopositive cells and puncta was qualitatively similar to that which we have described previously. However, in the neonatally-lesioned mice barrel rows B and C were not visible in the right hemisphere sections and there was a marked reduction in GABA-IR, with fewer immunopositive cells. Many of those that did show GABA-IR stained only weakly and the puncta were also fewer in number than in the left (control) hemisphere where the GABA-IR pattern and staining intensity was normal.

Cholinergic markers in the plasticity of murine barrel field.

Cholinergic muscarinic receptor binding and acetylcholinesterase (AChE) histochemistry were studied in the barrel cortex of adult, vibrissae deprived and vibrissae denervated mice. In the control barrel field muscarinic receptors labeled with [3H]quinuclidinyl benzilate ([3H]QNB) showed a higher density in the granular cortex and a higher accumulation of label in the barrels. AChE staining revealed a punctuated pattern corresponding to the barrels in the upper part of layer IV and a reverse-image pattern of staining showing only the walls of barrels in the lower part of layer IV. Neonatal denervation of rows of vibrissae lowered both binding of [3H]QNB to the tissue in the shrunken rows and AChE activity in the denervated rows of barrels. Deprivation and late denervation produced no effects on either pattern, or intensity, of [3H]QNB labeling and AChE staining. These observations suggest that the changes in cholinergic markers are related to the altered morphological structure and not to the abnormal functioning of the barrel cortex which received reduced sensory input from the vibrissae.