KCC2 Manipulation Alters Features of Migrating Interneurons in Ferret Neocortex.

KCC2 is a brain specific chloride-potassium cotransporter affecting neuronal development including migration and cellular maturation. It modulates chloride homeostasis influencing the switch of GABA from depolarizing to hyperpolarizing, which contributes to the cues that influence the termination of neuronal migration. The expression of KCC2 during migration of interneurons, therefore, correlates with the ability of these cells to respond to GABA as a stop signal. Manipulation of KCC2 in development can affect various aspects of migrating neurons, including the speed. We describe the effect of KCC2 downregulation and inhibition on features of migrating interneurons of normal ferret kits and those treated with methylazoxymethanol acetate, which increases KCC2. Treatment of organotypic cultures with Bisphenol A, an environmental toxin that alters gene expression, also downregulates KCC2 protein. In organotypic slices treated with the KCC2 antagonist VU0240551, chloride imaging shows inhibition of KCC2 via blockade of chloride flux. Time-lapse video imaging of organotypic cultures treated with either drug, shows a significant increase in the average speed, step size, and number of turns made by migrating neurons leaving the ganglionic eminence. Our findings demonstrate the harmful effect of environmental toxins on brain development and potential consequences in the pathogenesis of neurodevelopmental disorders.

Population based MRI and DTI templates of the adult ferret brain and tools for voxelwise analysis.

Non-invasive imaging has the potential to play a crucial role in the characterization and translation of experimental animal models to investigate human brain development and disorders, especially when employed to study animal models that more accurately represent features of human neuroanatomy. The purpose of this study was to build and make available MRI and DTI templates and analysis tools for the ferret brain as the ferret is a well-suited species for pre-clinical MRI studies with folded cortical surface, relatively high white matter volume and body dimensions that allow imaging with pre-clinical MRI scanners. Four ferret brain templates were built in this study - in-vivo MRI and DTI and ex-vivo MRI and DTI - using brain images across many ferrets and region of interest (ROI) masks corresponding to established ferret neuroanatomy were generated by semi-automatic and manual segmentation. The templates and ROI masks were used to create a web-based ferret brain viewing software for browsing the MRI and DTI volumes with annotations based on the ROI masks. A second objective of this study was to provide a careful description of the imaging methods used for acquisition, processing, registration and template building and to demonstrate several voxelwise analysis methods including Jacobian analysis of morphometry differences between the female and male brain and bias-free identification of DTI abnormalities in an injured ferret brain. The templates, tools and methodological optimization presented in this study are intended to advance non-invasive imaging approaches for human-similar animal species that will enable the use of pre-clinical MRI studies for understanding and treating brain disorders.

Altered migratory behavior of interneurons in a model of cortical dysplasia: the influence of elevated GABAA activity.

Appropriate function of the neocortex depends on timely generation and migration of cells produced in the germinal zones of the neocortex and ganglionic eminence (GE). Failure to accurately complete migration results in cortical dysplasia, a developmental syndrome implicated in many neurologic disorders. We developed a model of cortical dysplasia in ferrets involving administration of methylaxozymethanol acetate (MAM), an antimitotic, to pregnant ferrets on gestational day 33, leading to dramatic reduction of layer 4 in the neocortex. Here, using time-lapse video imaging, we investigate dynamic behavior of migrating cells arising from the GE and cortical ventricular zone (CVZ) in ferrets and the role of GABAA activity. Treatment with MAM significantly reduced migration speed and the relative proportion of cells arising from the GE demonstrating exploratory behavior. To a lesser extent, the behavior of cells leaving the CVZ was affected. Pharmacologic inhibition of GABAA receptors (GABAAR) improved the speed of migration and exploratory ability of migrating MAM-treated cells arising from the GE. Additionally, the expression of α2 and α3 subunits of GABAAR and the potassium chloride co-transporter (KCC2) increased in the neocortex of MAM-treated animals. After MAM treatment, increases in endogenous KCC2 and GABAAR combine to alter the dynamic properties and exploratory behavior of migrating interneurons in ferrets. We show a direct correlation between increased GABAA and KCC2 expression with impaired migration and ability to explore the environment.

Targeted disruption of layer 4 during development increases GABAA receptor neurotransmission in the neocortex.

Cortical dysplasia (CD) associates with clinical pathologies, including epilepsy and mental retardation. CD results from impaired migration of immature neurons to their cortical targets, leading to clustering of neural cells and changes in cortical properties. We developed a CD model by administering methylazoxymethanol (MAM), an anti-mitotic, to pregnant ferrets on embryonic day 33; this leads to reduction in cortical thickness in addition to redistribution and increased expression of GABAA receptors (GABAAR). We evaluated the impact of MAM treatment on GABAAR-mediated synaptic transmission in postnatal day 0-1 neurons, leaving the ganglionic eminence (GE) and in layer 2/3 pyramidal cells of postnatal day 28-38 ferrets. Embryonic day 33 MAM treatment significantly increases the amplitude and frequency of spontaneous GABAAR-mediated inhibitory postsynaptic currents (IPSCs) in the cells leaving the GE. In older MAM-treated animals, the amplitude and frequency of GABAAR-mediated spontaneous IPSCs in layer 2/3 pyramidal cells is increased, as are the amplitude and frequency of miniature IPSCs. The kinetics of GABAAR opening also altered following treatment with MAM. Western blot analysis shows that the expression of the GABAAα3R and GABAAγ2R subunits amplified in our model animals. We did not observe any significant change in the passive properties of either the layer 2/3 pyramidal cells or cells leaving the GE after MAM treatment. These observations reinforce the idea that synaptic neurotransmission through GABAAR enhances following treatment with MAM and coincides with our finding of increased GABAAαR expression within the upper cortical layers. Overall, we demonstrate that small amounts of toxins delivered during corticogenesis can result in long-lasting changes in ambient expression of GABAAR that influence intrinsic neuronal properties.

Transplants of NGF-secreting fibroblasts restore stimulus-evoked activity in barrel cortex of basal-forebrain-lesioned rats.

Cholinergic nuclei in the basal forebrain supply the cerebral cortex with acetylcholine (ACh). Depletion of cholinergic fibers following basal forebrain lesion results in reduced stimulus-evoked functional activity in rat barrel cortex in response to whisker stimulation. We showed previously that exogenous delivery of nerve growth factor (NGF) to the lateral ventricle restores reduced functional activity toward normal despite persistent reductions in cortical cholinergic activity. Gene transfer of therapeutic peptides using genetically engineered cells allows for localized and biological delivery of compounds to the CNS, circumventing systemic administration or repetitive invasive surgery. In this study, we grafted genetically engineered fibroblasts that secrete NGF (NGF+) into three CNS loci of rats with unilateral basal forebrain lesions, along with control fibroblasts (NGF-) that did not secrete NGF. Only NGF+ fibroblasts grafted into ACh-depleted somatosensory cortex resulted in improvement of functional activity following cholinergic depletion. NGF+ fibroblast transplants into the lateral ventricle or basal forebrain did not improve functional activity nor did NGF- fibroblasts in any site. Similar to our previous experiments using intraventricular NGF injections, despite improvements in functional activity, the affected barrel cortex remained depleted of acetylcholinesterase-stained fibers following insertion of NGF+ fibroblasts. These data support the idea that NGF can act directly on the cerebral cortex following reductions in cholinergic innervation. The mechanism of NGF action is elusive, however, since the presence of its high-affinity receptor, trkA, in the cerebral cortex is controversial.

Laminar specific alterations of thalamocortical projections in organotypic cultures following layer 4 disruption in ferret somatosensory cortex.

The developing neocortex influences the growth of thalamocortical projections. Layer 4 in particular receives the majority of input from the thalamus and is important in instructing thalamic afferents to terminate. Previous in vivo experiments demonstrated that disruption of layer 4 during corticogenesis in ferret somatosensory cortex by application of methylazoxy methanol acetate (MAM) prevents proper termination of thalamic afferents in appropriate cortical regions. To further explore the role of layer 4 in thalamocortical development, we prepared organotypic cocultures consisting of normal gestational day 0 (P0) ferret thalamus paired with normal, embryonic day 33 (E33), or E38 MAM-treated cortex obtained from ferrets at either P0 or P7. Injection of MAM on E33 disrupts layer 4 formation, whereas similar injections on E38 interfere with layer 2 formation. The cocultures grew together for a number of days, then discrete injections of either fluorescent dextrans or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI) were made into the thalamic piece. The labeled thalamic afferents that grew into the cortical slice were analysed and the sites of their terminations quantified after 3, 5, or 7-10 days in culture (DIC). Our results varied somewhat with the amount of time in culture, but the preponderance of thalamic fibers in normal cortex terminated in layer 4, whereas their counterparts in E33 MAM-treated cortex grew beyond the cortical plate and many fibers terminated inappropriately within lower cortical layers or white matter. Terminal distribution of thalamic fibers in E38 MAM-treated cortex looked similar to normal. These results demonstrate that the cells of layer 4 provide thalamic afferents with important positional and termination cues.

Disruption of layers 3 and 4 during development results in altered thalamocortical projections in ferret somatosensory cortex.

The precision of projections from dorsal thalamus to neocortex are key toward understanding overall cortical organization and function. To identify the significance of layer 4 cells in receiving the bulk of thalamic projections in somatosensory cortex, we disrupted layer 4 genesis and studied the effect on thalamic terminations in ferrets. Second, we ascertained the result of layer 4 disruption on functional responses and topographic organization. Methylazoxy methanol (MAM) was injected into pregnant ferrets on embryonic day 33 (E33), when most layer 4 neurons of somatosensory cortex are generated. This treatment resulted in dramatic reduction in the thickness of targeted layer 4. E38 MAM treatment was used as a control, when layer 2-3 neurons are generated. The projections of ventrobasal thalamus into somatosensory cortex were studied using DiI injections. We found only subtle differences between groups (normal, E33, or E38 MAM-treated) in the thalamic afferent pattern on postnatal day 1 (P1) and P7. On P14, thalamic terminations distribute almost equally throughout the remaining cortical layers in the E33 MAM-treated group compared with normal and E38 MAM-treated animals, in which the ventrobasal thalamus projects primarily to central layers. Electrophysiological recordings conducted on mature ferrets treated with MAM on E33 demonstrated that somatotopic organization and receptive field size are normal. These findings emphasize the importance of layer 4 in determining the normal laminar pattern of thalamic termination and suggest that, although its absence is likely to impact on complex neocortical functional responses, topographic organization does not arise from the influence of layer 4.

Nerve growth factor increases stimulus-evoked metabolic activity in acetylcholine-depleted barrel cortex.

Lesions of the basal forebrain deplete the neocortex of cholinergic fibers. Acetylcholine depletion in the somatosensory cortex of rats results in reduced stimulus-evoked activity in response to whisker stimulation. Previous studies demonstrate that embryonic basal forebrain transplants improve functional activity toward normal. It is not clear if the activity increase is due to cholinergic replacement or other factors present in the graft. In this study, we examined the possibility that nerve growth factor (NGF), a neurotrophin known as a survival factor and a specific protectant for cholinergic basal forebrain neurons, can preserve basal forebrain cells after a lesion and restore functional activity in the somatosensory cortex. We report that NGF alone is capable of restoring functional activity in the barrel cortex of animals with basal forebrain lesions, while vehicle injections of saline do not alter activity. Both high (10 microg) and low (5 microg) doses of NGF unilaterally injected into the lateral ventricle improved stimulus-evoked functional activity during bilateral whisker stimulation. The mechanism of NGF action is not clear since the restoration of functional activity in cortex was not accompanied by increased cholinergic activity as detected by acetylcholinesterase fiber staining. NGF may act directly on cortical neurons, although its site of action is not well defined.

Anatomical correlates of representational map reorganization induced by partial vibrissectomy in the barrel cortex of adult mice.

We examined the potential for changes in cortical connectivity to accompany long-term plastic changes in functional cortical representations of mystacial vibrissae. Plasticity in the barrel cortex of young adult mice was evoked by vibrissectomy that spared row C of whiskers. We found that 2-deoxyglucose brain mapping causes a progressive expansion of cortical representation of the spared vibrissae. Two months after vibrissectomy, when the width of the cortical map of the spared row of vibrissae doubled, living cortical slices of the barrel cortex were injected with fluorescent dextrans. The injections were centered on spared, deprived and control vibrissal columns. The injections labeled three intracortical projection systems: (i) local connections from one vibrissal column to neighboring columns; (ii) long-range projections running in the septa and walls of the barrels and spanning several barrels; and (iii) very-long-range fibers running horizontally in the lower part of layer V. The local, short-range projection system was analysed following small injections into the centers of columns in layers III and IV. We found that injections into spared barrels labeled axons extending for significantly greater distances in all layers (except layer V), and labeled cell bodies situated significantly further, than after injections into deprived or control barrels. Also, the total axonal density labeled by injections into the spared barrel was higher by 70% than for the deprived or control barrels. Alterations of topographical maps in adult somatosensory cortex may occur immediately after functional denervation, but may also increase with time, as in the case of our experimental situation. Our results indicate that persistent, long-term plastic change can remodel connectivity in the barrel cortex.

Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers.

Early generated layers of neocortex are important factors in forming the subsequent architecture of the cerebral cortex. To further explore the role of early generated cortex, we disrupted formation of an early generated cohort of cells by intraperitoneal injections of the mitotic inhibitor methylazoxymethanol (MAM) into pregnant ferrets timed to coincide with generation of subplate neurons in the ventricular zone. Our studies demonstrate that if early development of the neocortex is interrupted by injection of MAM during embryogenesis (on embryonic day 24 or 28; E24 or E28), a distinct laminar pattern fails to form properly in the parietal cortex. A reduced number of MAP2-positive cells were observed in the region of the subplate when compared with the number of MAP2-positive cells found in normal animals. Interference with the superficial neocortical layers that form later during development (on embryonic day 33) by appropriately timed MAM injections does not result in a severely disrupted laminar pattern. The interrupted laminar pattern that arises after early MAM injections coincides with distorted radial glial cells (identified by immunoreactivity to the intermediate filament protein, vimentin), which occur after early, but not late, MAM injections. Further analysis suggests that interference with early development of neocortex leads to premature differentiation of radial glial cells into astrocytes, as demonstrated by the presence of glial fibrillary acidic protein (GFAP). Experiments involving injections of the thymidine analog, bromodeoxyuridine (BRDU), demonstrated that 4 days after E24 MAM injection cells are generated and migrate into the thin cortical plate. By E38, however, cells continue to be generated in animals treated with MAM on E24 but do not reach their normal positions in the cortical plate. In addition, immunoreactivity using the CR50 antibody, which identifies presumptive Cajal-Retzius cells present in layer 1, demonstrates that the CR50-positive cells, normally precisely located in the outer portion of layer 1, are distributed in disarray throughout the thickness of the neocortex and intermediate zone in early MAM-treated animals, but not in those treated with MAM injections later during gestation. These findings are consistent with the idea that early generated layers are important in providing factors that maintain the environment necessary for subsequent neuronal migration and formation of neocortical layers.

Organization of the forepaw representation in ferret somatosensory cortex.

Little is known about the function and structure of ferret somatosensory cortex. We used anatomical methods and multi-unit recordings to characterize the cytoarchitecture, functional responses and topography of the forepaw representation in ferret somatosensory cortex. The representation of the cutaneous ferret forepaw encompasses approximately the caudal half of the posterior sigmoid gyrus. The posterior sigmoid gyrus and coronal sulcus contain unique cytoarchitectonic fields that conform in large part to earlier descriptions of somatosensory regions in the cat. The cytoarchitectonic regions form mediolateral bands that comprise areas 4, 3a, 3b, 1, and 2 (from rostral to caudal). We studied most extensively areas 3a and 3b for functional responses to somatic stimuli; our data indicate that ferret somatosensory cortex contains at least two representations of the forepaw in these two areas. Our data also suggest that within ferret somatosensory cortex, the morphological and submodality features gradually, rather than abruptly, distinguish themselves as unique cortical fields.

Histogenesis of ferret somatosensory cortex.

The ferret has emerged as an important animal model for the study of neocortical development. Although detailed studies of the birthdates of neurons populating the ferret visual cortex are available, the birthdates of neurons that reside in somatosensory cortex have not been determined. The current study used bromodeoxyuridine to establish when neurons inhabiting the somatosensory cortex are generated in the ferret; some animals also received injections of [3H]thymidine. In contrast to reports of neurogenesis in ferret visual cortex, most neurons populating the somatosensory cortex have been generated by birth. Although components of all somatosensory cortical layers have been produced at postnatal day 0, the layers are not distinctly formed but develop over a period of several weeks. A small number of neurons continue to be produced for a few days postnatally. The majority of cells belonging to a given layer are born over a period of approximately 3 days, although the subplate and last (layer 2) generated layer take somewhat longer. Although neurogenesis of the neocortex begins along a similar time line for visual and somatosensory cortex, the neurons populating the visual cortex lag substantially during the generation of layer 4, which takes more than 1 week for ferret visual cortex. Layer formation in ferret somatosensory cortex follows many established principles of cortical neurogenesis, such as the well-known inside-out development of cortical layers and the rostro-to-caudal progression of cell birth. In comparison with the development of ferret visual cortex, however, the generation of the somatosensory cortex occurs remarkably early and may reflect distinct differences in mechanisms of development between the two sensory areas.

Development of intrinsic connections in cat somatosensory cortex.

We explored the development of clustered connections in cat somatosensory cortex by using intracortical injections of biocytin and carbocyanine dye (DiI). Biocytin injections in adults revealed clusters of retrogradely labeled cells, affirming earlier reports of the patchy nature of corticocortical connectivity in the adult cat somatosensory cortex. On postnatal days (PNDs) 1 and 3, a diffuse distribution of axons and labeled cells were found after DiI injections. A dramatic rearrangement of connections had taken place by PND 6. Well-defined clusters of labeled cells surrounded the injection site. Intercluster zones were relatively free of labeled cells and contained few labeled axons. At later ages (PNDs 12 and 15), a clear patchy distribution of intrinsic connections was seen. We analyzed neuronal clustering by using spatial point process analysis, which corroborated our qualitative observations. The density of labeled neurons was significantly higher in clusters than in the intercluster zones on PND 6. Autocorrelations run on profile plots of optical density values, along paths parallel to the edge of the injection sites (reflecting axonal distributions), revealed significant periodicity (P < .05; center-to-center approximately 750 pm) by PND 6. These data demonstrate that corticocortical connections in cat somatosensory cortex develop from a diffuse network at birth, which transforms into a patchy network by the end of the 1st postnatal week. This is substantially earlier than the development of local circuits in the cat visual cortex and may reflect fundamental differences in the organization of the two cortices.

Development of local connections in ferret somatosensory cortex.

Ferrets have become recognized as a useful and interesting model for study of neocortical development. Because of their immaturity at birth, it is possible to study very early events in the ontogeny of the brain. We used living slices of ferret somatosensory cortex to study the formation and development of intrinsic elements within the neocortex. A small number of fixed, hemisected brains injected with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) were also used. The slices were obtained from ferret kits aged postnatal day (P)1 to P62 and maintained in a chamber; each slice received injections of fluorescent-labeled dextrans. The injections were made at different ages in several distinct sites, which included the proliferative ventricular zone, the intervening white matter (or intermediate zone), and different sites of developing cortex, including the deeper cortical plate, which incorporated the subplate in young animals, and more superficial cortical sites depending on the age of the animal. Several animals also received injections into the ventrobasal thalamus. Injections into young animals (P1-7) produced a dominant radial pattern that extended from the ventricular zone into the cortex. Injections into the ventricular zone labeled many cells that appeared morphologically like radial glia as well as presumptive neurons. Although the predominant pattern was radial, injections in the ventricular zone often produced tangentially oriented cells and horizontally arranged fibers at the outer edge of the proliferative zone. These cells and fibers may provide a substrate for tangential dispersion of neurons within the neocortex. More superficial injections within the slice labeled lines of cells that appeared to be stacked upon one another in a radial pile in the cortex; the cortical plate received very few lateral projections. Data obtained from more mature slices indicated that although the overall pattern of staining remained radial, the precise character of the pattern changed to include more lateral spread into surrounding cortex, which eventually refined and developed into distinct patches by P28, when the overall cortical architecture appeared adult like. The data involving thalamocortical connections were more limited, but they indicated that the thalamus projects precisely to the somatosensory cortex in a point-to-point fashion from the earliest date studied (P0) and that the ventrobasal nucleus terminates upon the somatosensory cortex in a patchy manner during the early postnatal days of development. This study of the development of the somatosensory cortex confirms the ubiquitous nature of column-like connections throughout the neocortex and provides a novel view of the radial nature of early neocortical maturation.

The impact of basal forebrain lesions on the ability of rats to perform a sensory discrimination task involving barrel cortex.

Depletion of cortical acetylcholine (ACh) correlates with reduced stimulus-evoked 2-deoxyglucose (2-DG) uptake in rodent somatosensory cortex. We examined the effect of unilateral basal forebrain lesions and subsequent cortical ACh depletion on (1) the ability of rats to detect a passively applied deflection of the whiskers, and (2) whisker-evoked 2-DG uptake. Normal adult rats were trained on a T-maze to respond by turning in one direction if the whiskers were displaced and in the opposite direction on presentation of a sham stimulus; only the left set of whiskers was stimulated. When the animal performed at the 80% correct level for three consecutive sessions (criterion), it was randomly assigned to a group receiving either a saline injection (sham lesion) or an ibotenic acid injection (excitotoxic lesion) into the right basal forebrain. Behavioral testing continued until the animal returned to the prelesion criterion, at which time a terminal 2-DG experiment was conducted. None of the sham-lesioned rats experienced disruption of their ability to perform the task. All excitotoxic basal forebrain-lesioned rats were impaired in task performance, but eventually returned to prelesion performance levels. The length of time required to return to criterion was positively correlated with the amount of cortical ACh depletion. Despite the behavioral recovery of the ACh-depleted rats, 2-DG uptake in response to whisker stimulation continued to be reduced in the somatosensory cortex ipsilateral to the basal forebrain lesion. These findings suggest that ACh depletion, which results in a long-lasting decrease in neuronal responses to evoked stimuli, transiently impairs an animal's ability to perceive and appropriately respond to sensory information; the duration of impairment is related to the degree of depletion.

Long-term delayed vascularization of human neural transplants to the rat brain.

Human neural transplants are being developed to treat Parkinson's disease. Previous characterization of human transplants focused on neuronal development, while little is known of the interaction between the transplant and its environment, among which blood is of prime importance. We evaluated here the formation of blood vessels in human neural xenografts placed into the brain of rats immunosuppressed with cyclosporin A. Using capillary wall markers, we found that human transplants remain virtually nonvascularized for more than 1 month. Angiogenesis takes place very slowly and the density of blood vessels is still quite poor after 3 months, the fine structure of these capillaries, when they form, is apparently normal. Functional studies indicate that the vascular network formed in the transplant allows blood circulation and exhibits a working barrier to macromolecules. Glucose uptake and consumption and cytochrome oxidase activity are almost undetectable up to 3 months after grafting. These results demonstrate that vascularization is much delayed in human xenografts into the rat brain. This delay is likely to be dependent on the maturation of the transplanted tissue. A dedifferentiation of human endothelial cells cotransplanted with neural cells occurs since histochemical and immunocytochemical markers revealing endothelial cells in the human fetus are not present up to 1 month in the transplant. The origin of this phenomenon is a matter of speculation. How neural cells survive and mature in such conditions are issues of prime interest for the future of human neural grafting.

Developmental regulation of plasticity in cat somatosensory cortex.

1. The neocortical response to deprivation of somatic sensory input in young animals of different ages was compared with the same manipulation in adults. The response was measured through the use of 2-deoxyglucose (2DG) mapping. Although several features of the cortical response were similar in animals of all ages, the metabolic patterns evoked by somatic stimulation differed substantially from each other at all ages. 2. When adult cats receive a digit amputation and survive from 2 to 8 wk, the pattern of stimulus-evoked metabolic uptake expands dramatically in the somatosensory cortex contralateral to the deprived forepaw. Comparisons between the normal and experimental somatosensory cortices reveal that the distribution of activity on the experimental side was roughly an expanded version of the normal pattern. 3. Unilateral digit amputations of digit 2 were conducted on kittens 2, 4, or 6 wk old. They survived until 3-4 mo and then received a 2DG experiment, during which digit 3 was stimulated bilaterally. Evaluation of the evoked metabolic pattern indicated substantial differences from the activity elicited in adults undergoing identical manipulations. 4. The individual patches of activity that made up the metabolic pattern were similar in intensity in both hemispheres when the digit amputation was conducted at either 2, 4, or 6 wk. After a digit amputation at 2 wk, the patches were significantly narrower in the experimental hemisphere; after a digit amputation at 6 wk, the patches were significantly wider in the hemisphere receiving from the deprived forepaw. 5. Two-dimensional maps of 2DG uptake in areas 3b and 1 of the somatosensory cortex reveal that after a digit amputation at 2, 4, or 6 wk, the distribution of activity in the hemisphere receiving from the digit amputation was more dispersed and widespread than in the normal hemisphere. The dispersed pattern of uptake was not an expanded version of the normal pattern, but scattered over a wider region of somatosensory cortex. This observation is similar to the normal pattern of evoked activity seen in developing animals. 6. The total area of 2DG uptake in the somatosensory cortex contralateral to a digit amputation conducted at 2 or 4 wk was not greater than that in the normal hemisphere, even though it was more widespread. After a digit amputation at 6 wk, however, the area of evoked activity was greater in the experimental hemisphere but not of the magnitude as the same manipulation in an adult.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic basal forebrain transplants restore diminished metabolic activity in the somatosensory cortex of rats with acetylcholine depletion.

It has been known for several years that stimulus-evoked metabolic activity is reduced in the somatosensory cortex of animals with basal forebrain lesions that deplete the neocortex of acetylcholine (ACh). During 2-deoxyglucose (2-DG) experiments, animals with unilateral basal forebrain lesions demonstrate a decreased response to somatic stimulation, while background metabolic activity in the surrounding cortical regions remains normal. In an attempt to ameliorate these deficits, we examined the ability of embryonic cholinergic basal forebrain transplants inserted into neocortex to innervate surrounding cortical regions and restore functional 2-DG activity in adult host rats previously depleted of ACh by basal forebrain lesions. To accomplish this goal, a series of experiments were conducted in which we (1) depleted the cerebral cortex of ACh by injecting an excitotoxin into the rat basal forebrain, (2) transplanted embryonic basal forebrain or embryonic neocortical (control) tissue into the ACh-depleted cortex and, (3) 6-12 months later, used the 2-DG metabolic mapping technique to examine effects of the transplants on metabolic activity evoked by whisker stimulation in rat somatosensory (barrel) cortex. Histochemical analysis revealed that acetylcholinesterase (AChE) staining within 2 mm of the basal forebrain transplants was similar in density to the contralateral normal hemisphere. AChE staining farther than 2 mm from the basal forebrain transplants and throughout hemispheres containing neocortical (control) transplants was greatly reduced, with few AChE-positive fibers present, a finding typical of cerebral cortex in basal forebrain-lesioned animals. Stimulus-evoked 2-DG uptake in barrels adjacent to the basal forebrain transplants, and therefore within AChE-rich territory, was similar to that in corresponding barrels identically activated in the contralateral hemisphere. 2-DG activity was reduced, however, in stimulated barrels outside the region of dense AChE-positive staining, as well as in all activated barrels in hemispheres containing control transplants of embryonic neocortex. These results indicate that transplantation of cell suspensions containing embryonic cholinergic basal forebrain, but not neocortex, can ameliorate basal forebrain lesion-induced deficits in functional activity, and that the restoration of activity is influenced by proximity to the transplant.

Development of metabolic activity patterns in the somatosensory cortex of cats.

1. The development of cortical responses to somatic stimulation was studied in kittens 2-5 wk of age using the 2-deoxyglucose (2DG) technique. During the 2DG experiment each kitten received an innocuous intermittent vertical displacement stimulus to the forepaw. 2. The pattern of metabolic activity was substantially different in young animals compared with adults. In the individual autoradiographs of the 2-wk-old kittens stimulus-evoked 2DG uptake in primary somatosensory cortex was localized to a small spot in the upper portion of the cortex, whereas in the adult the label extended vertically through the cortical layers and appeared more column-like. Individual patches of label were substantially smaller and less dense in young animals. Over a period of several weeks the evoked activity evolved to the more extensive adult pattern. The 2DG uptake displayed a mature distribution by approximately 4-5 wk of age. During this period, the cortical architecture also evolved from an immature to a mature arrangement. 3. The evoked activity was reconstructed into two-dimensional maps; the distribution of label > or = 1.5 SD above background was considered to be stimulus related. In the adult, the pattern appeared as a strip or strips of increased metabolic activity that extended in the rostrocaudal direction for approximately 1 mm. In contrast, the activity pattern in animals 2-4 wk old was less discretely organized into "strips" and was more diffusely spread over several mms of somatosensory cortex. The two-dimensional pattern gradually coalesced into a more localized strip by approximately 4-5 wk of age. Although the pattern of label was more widespread in the young animals, the absolute distance of the spread of activity did not vary substantially, regardless of the age of the animal. 4. Other measurements regarding the distribution of activity at different ages indicate that the amount of cortex activated increases in absolute terms, although the percent of cortex activated by the stimulus decreases. The overall intensity of the 2DG uptake as measured on the two-dimensional maps increases with age, as does the variability of the 2DG uptake; a wider range of intensity values is seen in the adult. Plots created from the individual two-dimensional reconstructions allowed a measure of "patch strength" at different ages. These histograms relate the most intense region of uptake in a given map to the spatial distribution of activity spreading in the medial and lateral directions.(ABSTRACT TRUNCATED AT 400 WORDS)