The effect of autonomous alpha-CaMKII expression on sensory responses and experience-dependent plasticity in mouse barrel cortex.

The calcium/calmodulin kinase II (CaMKII) autophosphorylation site is thought to be important for plasticity, learning and memory. If autophosphorylation is prevented by a point mutation (T286A) LTP is blocked in the hippocampus and cortex. Conversely, if the point mutation mimics autophosphorylation (T286D) a range of frequencies that normally produce LTP in wild types cause LTD instead. In order to test whether the alphaCaMKII-T286D mutation increases levels of depression in vivo, we examined the effect of the alphaCaMKII-T286D transgene on plasticity induced in the barrel cortex by whisker deprivation. Surprisingly, the mutation did not affect depression or potentiation. However, in animals reared with the transgene turned on from birth, the surround receptive field responses were greater than normal. This effect may be due to the potentiating action of autophosphorylated CaMKII during early development.

The role of cortical activity in experience-dependent potentiation and depression of sensory responses in rat barrel cortex.

The role of cortical activity in experience-dependent cortical plasticity was studied in the rat barrel cortex. Plasticity was induced by depriving every other whisker in a chessboard pattern, which is known to cause depression of responses to deprived whisker stimulation and potentiation of responses to spared whisker stimulation. Postsynaptic activity was blocked by muscimol released from elvax slow-release polymer located under the dura and over the barrel field. Spared whisker responses potentiated 2.5-fold in layer II/III and 2.9-fold in layer IV of the near-neighbor barrel in animals implanted with saline-elvax. In contrast, in whisker-deprived animals implanted with muscimol-elvax, responses were indistinguishable from those in undeprived animals. Similarly, in the spared barrel itself, spared whisker responses potentiated 1.3-fold in layer IV in animals implanted with saline-elvax but not at all in muscimol-treated animals. Whiskers that were deprived and then allowed to regrow showed depressed responses in saline-elvax-treated animals, in which 40% of the cells in layer II/III and 26% in layer IV were unresponsive to their principal whisker. These values fell to 17 and 3% for layers II/III and IV, respectively, in muscimol-treated animals, and the response magnitude distributions were indistinguishable from undeprived cases. Cortical activity block had no acute effect on the ventroposteriomedial nucleus responses and had a transient facilitatory effect after 4 d of muscimol treatment, which returned to baseline as the muscimol treatment wore off. We conclude from these studies that cortical activity is required for potentiation and depression of sensory responses in barrel cortex.

Plasticity and stability of somatosensory maps in thalamus and cortex.

Work on elucidating the mechanisms of plasticity in somatosensory maps continues apace. Recent work has focused on both the nature of the thalamocortical interactions that determine plasticity, and on the differences between plasticity induced by nerve block or damage versus that induced by experience. Recordings from awake behaving animals have thrown light on the thalamocortical circuit mechanisms that underlie map plasticity; meanwhile, intracellular recordings from cortical slices have thrown light on the precise synaptic mechanisms underlying plasticity.

The role of alpha-CaMKII autophosphorylation in neocortical experience-dependent plasticity.

Calcium/calmodulin kinase type II (CaMKII) is a major postsynaptic density protein. CaMKII is postulated to act as a 'molecular switch', which, when triggered by a transient rise in calcium influx, becomes active for prolonged periods because of its ability to autophosphorylate. We studied experience-dependent plasticity in the barrel cortex of mice carrying a point mutation of the alpha-CaMKII gene (T286A), which abolishes this enzyme's ability to autophosphorylate. Plasticity was prevented in adult and adolescent mice homozygous for the mutation, but was normal in heterozygotes and wild-type littermates. These results provide evidence that the molecular switch hypothesis is valid for neocortical experience-dependent plasticity.

Age-dependent response of the mouse barrel cortex to sensory deprivation: a 2-deoxyglucose study.

The effect of age on the plastic response of vibrissal barrel cortex to deprivation was examined in adolescent (1 month at the start of the procedure, 2 months at testing) and mature (10- to 11-month-old) mice. A single vibrissa was plucked out for 3 weeks and allowed to regrow for 10 days; it was previously found that this deprivation paradigm induces strong downregulation of the deprived input. The results of deprivation were assessed with 2-deoxyglucose functional brain-mapping autoradiography. Deprivation was found to reduce the ability of the deprived vibrissa to activate the cortex both in adolescent and mature mice. However, while in young animals the decrease of the extent of cortical labeling, compared with the normal control, was observed in all examined cortical layers (II/III, IV, and V), in older mice the effect was reduced in layers II/III and absent in layer IV. The suppression of response of the infragranular layers was not affected by age. Transition from adolescent to mature adulthood brings about a layer-specific decline in depression of the cortical response to the deprived input.

Upregulation of cAMP response element-mediated gene expression during experience-dependent plasticity in adult neocortex.

Gene transcription is thought to be essential for memory consolidation and long-lasting changes in synaptic function. In particular, the signal transduction pathways that activate the transcription factor cAMP response element binding protein (CREB) have been implicated in the process of synaptic potentiation. To study the involvement of this pathway in neocortical plasticity within the barrel cortex, we have used a strain of mice carrying a LacZ reporter gene with six cAMP response elements (CREs) upstream of a minimal promoter. Removal of all but one facial whisker results in the expansion of the spared whisker's functional representation within somatosensory cortex. Under the same conditions of whisker deprivation, we observed a strong (eightfold compared with baseline) and highly place-specific upregulation of CRE-mediated gene transcription in layer IV of the spared whisker barrel. Reporter gene upregulation occurred rapidly after deprivation (16 hr) and was only observed under experimental conditions capable of inducing whisker response potentiation. LacZ expression in layer IV was accompanied by an increase in responsiveness of a subpopulation of layers II/III cells to spared whisker stimulation as determined by in vivo single-unit recording. Given that CREB is involved in the expression of plasticity in superficial layers (Glazewski et al., 1999), and yet CRE-mediated gene expression occurs in layer IV, it is likely that the molecular events initiating plasticity occur presynaptically to the cells that exhibit changes in their receptive field properties.

Impaired experience-dependent plasticity in barrel cortex of mice lacking the alpha and delta isoforms of CREB.

The transcription factor cyclic-AMP response element binding protein (CREB) has been implicated in long-term plasticity processes in vertebrate and invertebrate species. In the absence of the alpha/delta CREB isoforms, performance is impaired in long-term memory tasks and the long-term maintenance of long-term potentiation (LTP) is impaired in the hippocampus. However, it is not known whether CREB plays a role in neocortical plasticity. Antibodies to CREB revealed that CREB-immunoreactive nuclei are present in all cortical layers but are more numerous in layers II/III, where they composed at least two-thirds the total population of cells. CREB-immunopositive cells were therefore present and densest in the very cortical layers that exhibit experience-dependent plasticity at this age. In order to assess the role of CREB in neocortical plasticity, we studied the effect of vibrissae deprivation on receptive field plasticity in the barrel cortex of mutant mice lacking the alpha/delta isoforms of CREB. A single vibrissa was spared and the others removed for 18 days. In wild-types this caused potentiation of the spared vibrissa response. However, in adult mutants (>6 months) spared vibrissa responses from homozygotes were potentiated less than in any adolescent animals or in adult wild-type littermates. Surround receptive field responses were abnormally large in homozygotes and failed to increase by the same amount as they did in wild-types. In contrast, the alpha/delta CREB mutation had no discernible effect on plasticity in cortical layers II/III of the younger adolescent age group (1-2 months), suggesting that different plasticity processes may operate at this age. Further tests showed that the beta isoform of CREB was up-regulated in the barrel cortex of the alpha/delta CREB knock-outs, suggesting that this subunit may have compensated partly for the loss of the alpha/delta isoforms. These studies suggests that CREB plays a role in experience-dependent plasticity in the adult neocortex.

Experience-dependent depression of vibrissae responses in adolescent rat barrel cortex.

A short period of vibrissae deprivation in an adolescent (approximately 1 month old) rat can lead to depression of the cortical response to stimulation of the regrown vibrissae. In a barrel column representing the deprived vibrissa, depression is greater for neurons located close to the barrel column representing the spared vibrissa. One possible explanation is that the spared vibrissa produces heterosynaptic depression of the principal vibrissa response (Glazewski & Fox, 1996). To test this idea further, we compared the effect of depriving all vibrissae (no heterosynaptic influence at all) with depriving a single vibrissa (maximal heterosynaptic influence expected). In addition we tested the origin of the depression by recording from subcortical structures. After 7 days' deprivation and 6-8 days' regrowth, we tested the responses of barrel cortex cells, thalamic VPm neurons and trigeminal ganglion cells to stimulation of the regrown vibrissae. We found that depression was greater in cortex if a single vibrissa had been deprived than if all vibrissae had been deprived. (Average principal vibrissae responses in single vibrissae deprived animals were 36% of those in all vibrissae deprived animals for layer II/III and 41% for layer IV.) This implicates the spared vibrissae in actively down-regulating responses to the deprived vibrissae. However, some depression could also be produced in animals deprived of all vibrissae (layers II/III were 39% and layer IV 74% of control levels). These results indicate that simple withdrawal of activation has a depressive effect on responses but that depression is far greater if some active inputs remain. Neither form of deprivation had an effect on responses to principal vibrissa stimulation in the thalamus or trigeminal ganglion however, suggesting that depression originates in the cortex. Within the cortex, intracortical connections seem most affected as the greatest depression was found in layers II/III and in layer IV among cells responding at intermediate latencies (9-14 ms).

Long-term potentiation in vivo in layers II/III of rat barrel cortex.

Long-term potentiation was studied in vivo in the rat barrel cortex. It was found that LTP lasting several hours could be induced in layer II/III by tetanic stimuli applied in layer IV. The probability of inducing LTP at a given site was high (86%) provided that the electrodes were not displaced too far horizontally. LTP was not observed if the stimulating electrode was located on the far side of the neighbouring barrel-column from the recording electrode. The strongest LTP was induced by stimulating layer IV septal locations or the edge of the barrel and recording in the near half of the neighbouring barrel. However, examples were found of LTP from layer IV to II/III within the same barrel, within the same septum and from barrel to adjacent septum. The probability of inducing LTP on a particular occasion was greatly increased by iontophoresis of bicuculline at the recording site during the tetanus (from 20 to 55% judged by a change in peak amplitude). The average increase in the peak amplitude was 29 +/- 3.2% for protocol 1 (urethane anesthesia, monopolar stimulation) and 23 +/- 7% for protocol 2 (barbiturate anesthesia, bipolar stimulation). The probability of inducing LTP was greater if the first tetanus was accompanied by BMI application (67%) than for any subsequent attempts (39%). These results suggest it should be possible to study the effect of LTP on sensory processing in defined positions within the barrel field.

Experience-dependent changes in vibrissae evoked responses in the rodent barrel cortex.

A change in vibrissae complement in rodents leads to long-term changes in vibrissae dominance. These changes involve both potentiation of spared vibrissae responses and suppression of deprived vibrissae responses in adolescent animals. In adult animals only potentiation of spared vibrissae responses was detected. Suppression exhibits hetero- and homosynaptic components and appears to be cortical in origin, as is potentiation. The time course for potentiation and suppression in the barrel cortex of adolescent rats is different, with suppression preceeding potentiation by at least one week. There seems to be no critical period for potentiation in superficial layers of barrel cortex, but there is a critical period for suppression. Suppression cannot be evoked if plasticity is induced later than at 6 months of age nor maintained if experimental manipulations begin later than at three months. The molecular mechanisms that underlie plastic changes in the barrel cortex still remain unclear, although alpha-CamKII and to lesser extend alpha/beta-CREB appear to be involved.

Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex.

The segregation of thalamocortical inputs into eye-specific stripes in the developing cat or monkey visual cortex is prevented by manipulations that perturb or abolish neural activity in the visual pathway. Such findings show that proper development of the functional organization of visual cortex is dependent on normal patterns of neural activity. The generalisation of this conclusion to other sensory cortices has been questioned by findings that the segregation of thalamocortical afferents into a somatotopic barrel pattern in developing rodent primary somatosensory cortex (S1) is not prevented by activity blockade. We show that a temporary block of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat S1 during the critical period for barrel development disrupts the topographic refinement of thalamocortical connectivity and columnar organization. These effects are evident well after the blockade is ineffective and thus may be permanent. Our findings show that neural activity and specifically the activation of postsynaptic cortical neurons has a prominent role in establishing the primary sensory map in S1, as well as the topographic organization of higher order synaptic connections.

Time course of experience-dependent synaptic potentiation and depression in barrel cortex of adolescent rats.

1. Plasticity could be induced in (S1) barrel cortex of adolescent rats by reducing the complement of vibrissae on one side of the muzzle to a single whisker for a period of 7, 20, or 60 days. The effect of deprivation was assessed by quantitatively by measuring cortical responses to stimulation of the spared and regrown deprived vibrissae. Vibrissa responses were evoked using a standard stimulus generated by an electromechanical stimulator and measured using poststimulus time histogram analysis. 2. Cells located in layers II/III were found to be plastic beyond postnatal day 28 (P28), whereas cells located in layer IV were not. The vibrissa dominance distribution was shifted significantly toward the spared vibrissa after 7, 20, and 60 days of deprivation for cells located in layers II/III of barrel columns surrounding the D1 column (P < 0.0001, 2-factor analysis of variance). The vibrissa dominance distribution did not shift significantly for cells located in layer IV of surrounding barrels for any of the durations of deprivation tested (P > 0.1). After 7 days of deprivation, 37% of the cells located in layers II/III of the columns deprived vibrissae showed greater responses to the spared vibrissae than to their deprived principal vibrissa, compared with 11% in normally reared adolescent animals and 3% in adults. The percentage of cells dominated by the spared vibrissa was 65% after 20 days of deprivation and 43% after 60 days. 3. For cells located in layers II/III, short-term deprivation (7 days) caused a decrease in the absolute magnitude of response to stimulation of the deprived vibrissa (reduction to approximately 28% of control levels). However, no change could be detected in the spared (D1) vibrissa input to the same deprived columns. Therefore the increase in D1 dominance registered in the deprived columns was mainly due to a decrease in principal vibrissa response and no change in the spared D1 vibrissa response. 4. The first increase in spared vibrissa response was seen after 20 days of deprivation. The response magnitude cells located in layers II/III increased to 70% above control levels. Responses to deprived vibrissa stimulation were depressed at 20 days of deprivation (reduction to 35% of control), implying that the vibrissa dominance shift at 20 days was due to both an increase in spared vibrissa response and a decrease in deprived vibrissa response. 5. The spared vibrissa response was increased after 60 days of deprivation (110% above control) in both near and far halves of the barrel columns surrounding D1. On average, the deprived vibrissa response was depressed at 60 days (84% of control), although less than at 20 or 7 days, because of recovery of responsiveness in the far half of the deprived barrel column. Layer II/III cells located in the half of the barrel column farthest from the spared D1 barrel column showed normal levels of deprived vibrissa input, whereas cells located in the half of the barrel column closest to the spared D1 barrel column still exhibited depressed levels of deprived vibrissa input (54% of control). 6. Control experiments suggested that depression of the deprived vibrissa response could not be explained by nonspecific effects. Depression was not a function of animal's age, because normally reared P28 and adult animals showed similar principal vibrissa response levels. It was not a result of nonspecific depression of cortical responses. because the decreased response only occurred in cells of deprived barrel columns and not spared barrel columns (recorded in the same animals). Depression was not due to altered vibrissa mechanics, because the spared vibrissa response was similarly depressed in animals in which the vibrissae had been trimmed rather than removed. Finally, depression was input specific at 7 and 20 days, because only the deprived vibrissa responses were depressed, whereas spared vibrissa responses were either at control levels or at elevated levels for the same cells. 7.

Requirement for alpha-CaMKII in experience-dependent plasticity of the barrel cortex.

The mammalian sensory neocortex exhibits experience-dependent plasticity such that neurons modify their response properties according to changes in sensory experience. The synaptic plasticity mechanism of long-term potentiation requiring calcium-calmodulin-dependent kinase type II (CaMKII) could underlie experience-dependent plasticity. Plasticity in adult mice can be induced by changes in the patterns of tactile input to the barrel cortex. This response is strongly depressed in adult mice that lack the gene encoding alpha-CaMKII, although adolescent animals are unaffected. Thus, alpha-CaMKII is necessary either for the induction or for the expression of plasticity in adult mice.

Mechanisms underlying experience-dependent potentiation and depression of vibrissae responses in barrel cortex.

Plasticity was studied in the barrel cortex of rats and mice. Vibrissae deprivation in adult and adolescent animals caused changes in the response properties of cortical neurons to stimulation of spared and deprived vibrissae. Plasticity involved both potentiation of spared vibrissae responses and depression of deprived vibrissae responses in layer II/III of the cortex. Vibrissae response potentiation was found to require alpha CaMKII in adult cortex. Vibrissae response depression was not found to occur in adult cortex but was found in adolescent animals for the principal vibrissa. Preliminary results suggested that vibrissae response depression exhibits both hetero- and homosynaptic components.

NMDA receptors in mouse barrel cortex during normal development and following vibrissectomy.

The development of N-methyl-D-aspartate (NMDA) receptors and the effects of vibrissectomy upon [3H]MK-801 binding were examined in the barrel cortex of mice. Autoradiographic studies showed that initially very low binding of [3H]MK-801 sharply increased during the second postnatal week reaching the adult level by the end of the third week. Scatchard analysis performed on cortical membrane preparations indicated that this rise of [3H]MK-801 labelling was due to an increase in the number of binding sites and a decrease of Kd at postnatal day 15 and 28. The interlaminar differences of labelling were registered from postnatal day 8. Changes of interlaminar distribution were found during the second and third postnatal weeks. In adult barrel cortex the highest binding was found in supragranular layers. In layer IV of the cortex, the pattern of binding resembled the pattern of barrels. Unilateral denervation of vibrissae performed in neonatal and adult mice did not alter the intensity of [3H]MK-801 labelling or the laminar distribution of binding sites. These results suggest that NMDA receptor binding does not reflect the plastic changes occurring in the barrel cortex.

Effect of vibrissae deprivation on follicle innervation, neuropeptide synthesis in the trigeminal ganglion, and S1 barrel cortex plasticity.

Deprivation of vibrissae from an early age causes plasticity in S1 barrel cortex. This method of deprivation is most likely to induce plasticity by altering the balance of primary afferent activity from the deprived and spared vibrissae. To study whether or not induction or expression of this type of plasticity might be affected by follicle nerve injury caused by the deprivation technique, three different methods of detecting nerve injury were used: counting axon numbers in the distal follicle nerve, quantifying morphological changes in axons, and measuring neuropeptide expression in the trigeminal ganglion cells. First, nerves innervating follicles chronically deprived of vibrissae from birth had the same number of myelinated and unmyelinated axons as nerves from normally reared animals. Second, axons innervating deprived follicles showed no morphological changes in myelination or mitochondria characteristic of damaged nerves. Third, the corresponding nerve cell bodies in the trigeminal ganglion did not show upregulation of galanin or neuropeptide Y expression. In contrast, animals receiving mild injury of the follicle nerve endings (by cauterization of the follicle) showed profound changes in axonal myelination and mitochondria and increases in neuropeptide expression. These results imply that vibrissae deprivation does not act by inducing injury of the follicular nerve, suggesting that changes in the balance of follicle nerve activity are the cause of cortical plasticity. Consistent with this notion, a fourth experiment demonstrated that trimming the vibrissae induces cortical plasticity comparable to that induced by complete vibrissae removal.

On the role of NMDA receptors in blood pressure regulation in spontaneously hypertensive rats (SHR).

In the present study the binding of [(3)H]MK-801 to glutamatergic receptors of the NMDA type was compared in spontaneously hypertensive (SHR) and normotensive (WKY) rats in various brain structures (including nucleus tractus solitarii) by quantitative receptor autoradiography. Additionally, blood pressure changes after treatment with the NMDA antagonist MK-801 were studied in both strains. There were no differences between SHR and WKY rats either in the level of [(3)H]MK-801 binding or in the hypertensive reaction to MK-801.

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.

Development of NMDA receptor-channel complex and L-type calcium channels in mouse hippocampus.

In vitro binding autoradiography was used to examine the pattern and intensity of binding of [3H]glutamate to NMDA receptors, [3H]MK 801 to NMDA receptor associated channels, and [3H]PN-200 110 to L-type calcium channels in the hippocampus of mice aged 3-70 days. The distribution of NMDA receptors and NMDA receptor associated channels was similar but not identical at the tested ages. Beginning with postnatal day 8, high binding levels were confined mostly to the hippocampal strata: the oriens and radiatum (CA1 and CA3 with [3H]MK 801 labeling but only CA1 with NMDA displaced [3H]glutamate labeling), the moleculare (higher labeling with [3H]MK 801 than with NMDA displaced [3H]glutamate binding), and the lucidum. The binding values for NMDA receptor-channel complex rose in the examined period (especially within the second and third week), reaching a plateau at the end of the third postnatal week. Sharp growth of binding within the second and third week of life was about 50% greater with [3H]MK 801 than with NMDA displaced [3H] glutamate labeling. L-type calcium channels were found to be most abundant in the strata: the oriens of the CA3 field, the moleculare, and the lucidum. The time course of binding value changes for the calcium channel was similar to the time course found for the NMDA receptor-channel complex.

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.