Amyloid-beta expression in retrosplenial cortex of triple transgenic mice: relationship to cholinergic axonal afferents from medial septum.

Triple transgenic (3xTg-AD) mice harboring the presenilin 1, amyloid precursor protein, and tau transgenes (Oddo et al., 2003b) display prominent levels of amyloid-beta (Abeta) immunoreactivity in forebrain regions. The Abeta immunoreactivity is first seen intracellularly in neurons and later as extracellular plaque deposits. The present study examined Abeta immunoreactivity that occurs in layer III of the granular division of retrosplenial cortex (RSg). This pattern of Abeta immunoreactivity in layer III of RSg develops relatively late, and is seen in animals older than 14 months. The appearance of the Abeta immunoreactivity is similar to an axonal terminal field and thus may offer a unique opportunity to study the relationship between afferent projections and the formation of Abeta deposits. Axonal tract tracing techniques demonstrated that the pattern of axon terminal labeling in layer III of RSg, following placement of DiI in medial septum, is remarkably similar to the pattern of cholinergic axons in RSg, as detected by acetylcholinesterase histochemical staining, choline acetyltransferase immunoreactivity, or p75 receptor immunoreactivity; this pattern also is strikingly similar to the band of Abeta immunoreactivity. In animals sustaining early damage to the medial septal nucleus (prior to the advent of Abeta immunoreactivity), the band of Abeta in layer III of RSg does not develop; the corresponding band of cholinergic markers also is eliminated. In older animals (after the appearance of the Abeta immunoreactivity) damage to cholinergic afferents by electrolytic lesions, immunotoxin lesions, or cutting the cingulate bundle, result in a rapid loss of the cholinergic markers and a slower reduction of Abeta immunoreactivity. These results suggest that the septal cholinergic axonal projections transport Abeta or amyloid precursor protein (APP) to layer III of RSg.

A role for neurotrophin-3 in targeting developing cholinergic axon projections to cerebral cortex.

This study examined the relationship between expression of neurotrophin-3 (NT-3) and the ingrowth of cholinergic axonal projections in cerebral cortex. Patterns of expression of NT-3 (defined by beta-galactosidase reporter expression in heterozygous offspring of transgenic NT-3(lacZneo/+) mice) revealed that limbic cortical regions (including frontal, cingulate, and insular cortex, as well as the dentate gyrus) express NT-3 and that these cortical regions receive early and relatively dense cholinergic axons (stained for acetylcholinesterase, AChE). Using the dentate gyrus as a model system, studies revealed that expression of the NT-3 reporter parallels, and precedes by approximately 2 days, the ingrowth of AChE positive cholinergic axons. Studies of forebrain organotypic slice cultures demonstrate that basal forebrain-derived cholinergic axons extend into cortical regions in a pattern that mimics the pattern of expression of the NT-3 reporter. Similarly, chimeric co-cultures, combining wild type septum with a slice of hippocampus from heterozygous NT-3(lacZneo/+) mice, demonstrate that cholinergic axons grow into regions of the dentate gyrus that express the NT-3 reporter. Hemisphere slice cultures made from NT-3 knockout mice reveal cholinergic axonal growth into cortex, but these axons do not form the regional pattern characteristic of slice cultures made from wild type or heterozygous NT-3(lacZneo/+) mice. Further, chimeric co-cultures made using slices of wild type septum combined with slices of hippocampus from NT-3 knockout mice demonstrate robust cholinergic axonal growth into the hippocampus, but the cholinergic axons do not form the characteristic preterminal pattern associated with the dentate gyrus. Slice cultures from limbic cortical tissue from the NT-3 null mice do not display exaggerated levels of cell death. In aggregate, these data support the hypothesis that expression of NT-3 by cortical neurons serves to attract basal forebrain cholinergic projections to their target cells in cerebral cortex.

Chromatographic behavior of ion pair enantiomers of dansyl leucine cyclohexylammonium salt on a beta-cyclodextrin stationary phase and the effect of a competitive-binding mobile phase additive.

The separation of dansyl leucine enantiomers on a beta-cyclodextrin stationary phase is significantly complicated by the association of the amino acid with its cyclohexylammonium counter ion, in a mobile phase of 80:20 (v/v) methanol-water. This produces very unusual chromatography, with two partially superimposed peaks observed for each enantiomer at lower column temperatures. The peak shape is attributed to the irreversible, oncolumn conversion of the ion pair (I) to the free, protonated (neutral) dansyl amino acid (II+H). Increasing the ionic strength of the mobile phase greatly improves the chromatography by transforming the solute species to enantiomers of II (the anionic, free amino acid). Van't Hoff plots are constructed for both species I and II (under different mobile phase conditions) to provide thermodynamic insight into the major enantioselective driving forces of separation. The chiral discrimination of the stationary phase is found to be primarily enthalpically driven for both solutes. Finally, 1-adamantanecarboxylic acid (ACA) is investigated as a solute-competitive mobile phase additive to intentionally block the hydrophobic cyclodextrin cavities on the stationary phase. By varying the concentration of ACA additive in the mobile phase, control over the retention and chiral recognition of the stationary phase is demonstrated.

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation.

Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1-21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions. These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.

Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.

Possible target preferences of basal forebrain cholinergic neurons were studied in organotypic slice cultures. Cholinergic neurons in slices of medial septum or substantia innominata send axons into both hippocampus and neocortex when co-cultured together. However, septal cholinergic axons course through adjacent slices of neocortex to reach and branch densely in slices of hippocampus, but septal axons seldom grow beyond adjacent hippocampal tissue to reach neocortex. In contrast, cholinergic axons from substantia innominata commonly grow through hippocampus to reach neocortex, and also grow through neocortex to reach hippocampus, with similar branching densities in each target. The greater density of septal axonal branches in hippocampus than in neocortex suggests a preference of septal axons for the hippocampal target.

Thalamo-cortical afferents control transient expression of the dopamine D(3) receptor in the rat somatosensory cortex.

The D(3) dopamine receptor (D(3)R) is selectively and transiently expressed in the barrel neurons of the somatosensory cortex (SI) between the first and second postnatal weeks. The D(3)R expression starts after the initial ingrowth of thalamocortical afferents (TCAs) into the barrel cortex and could be induced or controlled by them. We show that unilateral electrolytic lesion of the thalamic ventrobasal complex immediately after birth leads to a decrease in the D(3)R mRNA concentration in the lesioned SI 7 days after the lesion, whereas the D(3)R binding is little affected. Fourteen days after the neonatal thalamic lesion, the D(3)R binding and mRNA are drastically reduced and the barrel-like pattern of the D(3)R is absent. Elevation of the D(3) binding normally seen between the first and second postnatal weeks does not occur. Thalamic lesion on P6 differentially affects the D(3)R expression. One day after the lesion, the D(3) binding and mRNA are down-regulated, but the effect is transient. Five days after the lesion the concentration of D(3) mRNA in the lesioned hemisphere returns to the control level. The typical barrel-like pattern of D(3)R expression is evident in the lesioned SI, although TCAs are completely absent. Quantitative analysis demonstrated elevated cellular levels of the D(3) mRNA in barrel neurons 5 days after the lesion. These higher levels are needed, perhaps, to support the increased production of the D(3)R protein appropriate for this age. Age-related dynamics of the D(3)R binding is retained in the lesioned SI, although the concentration of D(3)R sites remains reduced. These data demonstrate that intact thalamic input is essential for the formation of mechanisms responsible for developmental regulation of the D(3)R expression in the SI.

Arrival of afferents and the differentiation of target neurons: studies of developing cholinergic projections to the dentate gyrus.

This study examined the relationship between the development of cholinergic axons originating from the septum and a group of their target cells, the granule cells of the dentate gyrus of the rat. Acetylcholinesterase histochemistry was used to identify septal cholinergic afferents to the dentate gyrus; parallel studies used anterograde movement of a carbocyanine dye to label the septal projections. Septal cholinergic axons are present in the molecular layer of the internal blade of the dentate gyrus shortly after birth, but these axons do not reach the external blade until several days later. Results demonstrate that acetylcholinesterase positive septal axons grow into the external blade of the dentate gyrus only after the recently generated granule cells have coalesced to form a clearly defined layer. Results from studies using in situ hybridization techniques demonstrate that dentate gyrus granule cells express messenger RNAs for brain derived neurotrophic factor and for neurotrophic factor 3 shortly after formation of the granule cell layer. Ingrowth of septal cholinergic axons follows two days after the formation of the external blade of the dentate gyrus and the expression of neurotrophin messenger RNAs by the dentate granule cells. These data support the hypothesis that target cell development is a prerequisite for attracting the ingrowth of septal afferent axons.

The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Do subplate neurons comprise a transient population of cells in developing neocortex of rats?

Studies were undertaken to determine whether neurons of the subplate layer represent a transient or stable population of cells in developing neocortex of rat. The first set of studies sought to determine the fraction of subplate neurons that is lost during early postnatal development. The optical dissector method was used to analyze fluorescently stained material in animals the age of postnatal day 0 (P0) to P40. These results demonstrate a reduction of slightly less than half of the total number of subplate neurons from P0 to P40. Counts of labeled cells in littermates at varied ages after [(3)H]thymidine or BRDU treatment on gestational day 14 (G14 - birthdate of occipital subplate neurons) or G18 (birthdate of layers III-IV neurons) demonstrate loss of approximately 50% of neurons in the subplate layer between P0 and P40, somewhat greater than the loss of neurons from cortical layers III-IV. The second set of studies investigated whether subplate neurons display cellular atrophy during postnatal development. Analysis of subplate neurons injected intracellularly with Lucifer yellow in fixed slice preparations indicates no reduction in soma size, number of dendrites, or extent of dendritic fields of subplate neurons taken from animals age P0 to P60. The third set of studies investigated whether functional markers of subplate neurons are reduced during postnatal development. Analysis of tissue stained histochemically for cytochrome oxidase or acetylcholinesterase, or stained immunocytochemically for GABA, somatostatin, or neuropeptide Y, demonstrate a remarkable loss of expression of staining patterns from late gestational ages to P20. These data demonstrate that, although subplate neurons seem not to be a transient population of cells in the usual sense of being eliminated by cell death or structural atrophy, the loss of histochemical and immunocytochemical markers indicates that they may be a functionally transient population of cells.

Enhanced survival and morphological features of basal forebrain cholinergic neurons in vitro: role of neurotrophins and other potential cortically derived cholinergic trophic factors.

The present study examined survival- and growth-enhancing effects of cortical cells on basal forebrain cholinergic neurons (BFCNs) in culture and the degree to which endogenous nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) contribute to those trophic effects. When fetal (17 days of gestation) basal forebrain (BF) cells were grown for 5 days in coculture with cortical neurons, staining for acetylcholinesterase (AChE) showed a threefold increase in the number of BFCNs relative to BF cultures without cortex. Most of these labeled cells also displayed enhanced somatic, dendritic, and axonal growth. Coculturing cortical neurons with BF cells taken from postnatal animals produced similar results but with a somewhat greater degree of morphologic enhancement. Function-neutralizing antibodies to NGF, BDNF, and NT-3 were employed to determine whether they would block the trophic effects of cortical neurons on postnatal BFCNs. Although no significant changes in numbers or morphological features of AChE(+) neurons were observed with treatment with individual antibodies, cocultures treated with a combination of all three antibodies displayed fewer morphologically enhanced AChE(+) cells and more nonenhanced cells; the total number of AChE(+) neurons was not significantly changed. Treatment of pure BF cultures with exogenous NGF, BDNF, and NT-3 increased the number of AChE(+) neurons but did not reproduce the morphologic enhancement of cortical cells on BFCNs. These results suggest that neurotrophins by themselves can increase survival of postnatal BFCNs in culture and may work in concert with other unknown cortically derived factors to enhance BFCN morphologic differentiation. The unidentified cortical factors may also have strong survival-enhancing effects on BFCNs that are independent of the known neurotrophins.

Distinctive morphological features of a subset of cortical neurons grown in the presence of basal forebrain neurons in vitro.

Basal forebrain cholinergic neurons (BFCNs) provide the major subcortical source of cholinergic input to cerebral cortex and play an important role in regulating cortical activity. The present study examined the ability of BFCNs to influence neocortical neuronal growth by examining effects of the presence of BFCNs on certain cortical neurons grown under the controlled conditions of dissociated cell culture. Initial experiments demonstrated distinctive morphological features of a population of neurons (labeled with SMI-32, a monoclonal antibody to nonphosphorylated neurofilament proteins that labels pyramidal neurons in vivo) in cocultures containing basal forebrain (BF) and cortical cells. These neurons (large neurons immunoreactive for SMI-32 [SMI-32(+) neurons]) were characterized as having extensive axons, greater soma size, and more dendritic growth than did most SMI-32(+) neurons in the cultures. Staining for SMI-32 in cocultures in which the cortical neurons were labeled with a fluorescent marker before adding the BF cells indicated that virtually all large SMI-32(+) neurons were of cortical origin. Eliminating BFCNs with the selective cholinergic immunotoxin 192 IgG-saporin resulted in a >80% decrease in the number of large SMI-32(+) neurons, although causing little damage to other cells in the treated cultures; this suggests that survival or maintenance of large SMI-32(+) neurons may depend on ongoing trophic support from BFCNs. Thus, present findings suggest that BFCNs may provide powerful growth- and/or survival-enhancing signals to a subset of cortical neurons.

Neonatal treatment with 192 IgG-saporin produces long-term forebrain cholinergic deficits and reduces dendritic branching and spine density of neocortical pyramidal neurons.

The role of basal forebrain-derived cholinergic afferents in the development of neocortex was studied in postnatal rats. Newborn rat pups received intraventricular injections of 192 IgG-saporin. Following survival periods ranging from 2 days to 6 months, the brains were processed to document the cholinergic lesion and to examine morphological consequences. Immunocytochemistry for choline acetyltransferase (ChAT) and in situ hybridization for ChAT mRNA demonstrate a loss of approximately 75% of the cholinergic neurons in the medial septum and nucleus of the diagonal band of Broca in the basal forebrain. In situ hybridization for glutamic acid decarboxylase mRNA reveals no loss of basal forebrain GABAergic neurons. Acetylcholinesterase histochemistry demonstrates a marked reduction of the cholinergic axons in neocortex. Cholinergic axons are reduced throughout the cortical layers; this reduction is more marked in medial than in lateral cortical areas. The thickness of neocortex is reduced by approximately 10%. Retrograde labeling of layer V cortico-collicular pyramidal cells reveals a reduction in cell body size and also a reduction in numbers of branches of apical dendrites. Spine densities on apical dendrites are reduced by approximately 20-25% in 192 IgG-saporin-treated cases; no change was detected in number of spines on basal dendrites. These results indicate a developmental or maintenance role for cholinergic afferents to cerebral cortical neurons.

Calcium regulation of agonist binding to alpha7-type nicotinic acetylcholine receptors in adult and fetal rat hippocampus.

Quantitative autoradiography was used to compare the binding properties of alpha7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of 125I-alpha-bungarotoxin (125I-alpha-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of 125I-alpha-BTX binding, as well as alpha-cobratoxin and nicotine potency to displace 125I-alpha-BTX, did not change with age. Addition of Ca2+ to the assay buffer did not alter 125I-alpha-BTX binding, or alpha-cobratoxin inhibition of 125I-alpha-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca2+ on agonist affinity was dose-dependent, with an EC50 value of 0.25-0.5 mM. Ca2+ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal 125I-alpha-BTX binding sites are largely similar across age. Ca2+ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the alpha7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.

Specificity of attachment and neurite outgrowth of dissociated basal forebrain cholinergic neurons seeded on to organotypic slice cultures of forebrain.

Development and differentiation of basal forebrain-derived cholinergic neurons were studied using a new technique that combines dissociated cell cultures with organotypic slice cultures. Slices of cerebral cortex or entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic cultures on membranes. Dissociated cell suspensions of basal forebrain tissue, taken from rat or mouse fetuses at gestational day 15-17, were seeded on to the slice cultures. Combined cultures were maintained for two to 14 days in vitro. Cultures processed for acetylcholinesterase histochemical staining demonstrated that stained neurons display regional variation in attachment to the slice, with most attachment occurring on cortex and with no detectable attachment on the caudate-putamen. Regional differences in attachment occur between cortical areas, with medial (cingulate) cortex showing much denser cell attachment than lateral (parietal) cortex, and across cortical layers, with layer I and deep layers showing more attachment than middle cortical layers. Similar patterns were observed on slices from rat brain irrespective of whether rat or mouse dissociated cells were used. Tyrosine hydroxylase-stained dissociated cells from ventral midbrain displayed a different pattern of attachment, with prominent attachment to the caudate putamen and less apparent specificity of regional and cortical laminar attachment. Little evidence of neurite outgrowth occurred during the first two days in vitro, but by four days, acetylcholinesterase-positive basal forebrain cells displayed several short and thick neurites that appeared to be dendrites, and one long process that appeared to be an axon. By seven days in vitro, dendrites are well developed and the presumed axon has extended branches over wide areas of cortex. These studies revealed several different types of cell-tissue interaction. The degree of cell growth and differentiation ranged from robust growth when dissociated cells were seeded on to slice cultures of normal target tissue, to apparently no attachment or growth when cells were seeded on to non-target tissue. This combined technique appears to be a useful method for studies of specificity of cell attachment and patterns of neurite outgrowth.

Cholinergic neurons from different subdivisions of the basal forebrain lack connectional specificity for cerebral cortical target sites in vitro.

Basal forebrain cholinergic neurons send their axons to cerebral cortex in a topographically organized projection. Experiments tested the hypothesis that this topographic organization results from target preferences of the cholinergic neurons. Slices containing either medial septum or substantia innominata were grown in co-culture with slices of lateral neocortex and hippocampus. Cholinergic neurons from septum and from substantia innominata projected axons into neocortex and hippocampus, without obvious differences in pattern or density. These data suggest that basal forebrain cholinergic neurons can innervate any portion of the cerebral mantle.

Cultured basal forebrain cholinergic neurons in contact with cortical cells display synapses, enhanced morphological features, and decreased dependence on nerve growth factor.

Prior studies examining the dependence of basal forebrain cholinergic neurons (BFCNs) on nerve growth factor (NGF) for survival have reached differing conclusions depending on the experimental paradigm employed, suggesting the importance of environmental and developmental variables. The present study examined the NGF dependence of BFCNs and modulatory effects of target (cortical) neurons under the controlled conditions of dissociated cell cultures. Initial experiments found BFCNs (identified by using choline acetyltransferase immunocytochemistry) in pure basal forebrain (BF) cultures to be dependent on NGF between the 2nd and 4th week in vitro. During that developmental period, NGF deprivation for 3 days, induced by application of anti-NGF antibody, resulted in degeneration of over 80% of BFCNs, whereas at earlier or later times, BFCNs were largely resistant to NGF deprivation. When BF neurons were plated together with cortical neurons (as dissociated co-cultures), the BFCNs grew neuritic processes (labeled with acetylcholinesterase histochemistry) that appeared to specifically target cortical neurons; electron microscopy revealed that synapses formed between these cells. BFCNs in co-cultures were more resistant to NGF deprivation, were larger, and had much more extensive neuritic growth than BFCNs in pure BF cultures. The resistance of BFCNs to NGF deprivation provided by cortical neurons could be largely reproduced by addition of other trophic factors (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT3; neurotrophin 4/5, NT4/5; or glial-derived neurotrophic factor, GDNF) during NGF deprivation in pure BF cultures. These results suggest that developing BFCNs undergo a critical period requiring trophic influences that may be provided by NGF or other trophic factors, as well as unknown factors derived from cortical neurons.

Cholinergic innervation of cerebral cortex in organotypic slice cultures: sustained basal forebrain and transient striatal cholinergic projections.

Slices of entire forebrain hemispheres were taken from early postnatal rat pups and maintained as organotypic slice cultures. Basal forebrain cholinergic neurons, identified by histochemical staining for acetylcholinesterase, develop axons that grow rapidly into cerebral cortex. Ingrowth occurs by two routes: some axons course laterally from the basal forebrain region to reach lateral neocortex; others course dorsally from the septum to reach medial cortex. By one to two weeks in vitro, acetylcholinesterase-positive axons have extended throughout most of the cortical territory. In addition to basal forebrain cholinergic axons, the normally local circuit cholinergic neurons of the striatum also send axons into cerebral cortex. These striatum-derived axons can be distinguished from basal forebrain axons by their distinct morphological characteristics and by their different response to excision of the striatum or basal forebrain. Further, acetylcholinesterase-positive axons in cortex that originate from striatum appear to retract or degenerate after about one week in culture, while those from basal forebrain remain present and apparently healthy beyond two weeks. These data document the basal forebrain cholinergic ingrowth into cerebral cortex using this whole hemisphere slice culture system and also demonstrate different degrees of maintenance of cortical afferents that are derived from different subcortical sources.

Regulation of alpha7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents.

Distributions of alpha7 nicotinic acetylcholine receptor (nAChR) mRNA and [125]alpha-bungarotoxin (alpha-BTX) binding sites in the developing rat somatosensory cortex were characterized in relation to acetylcholinesterase (AChE) histochemical staining of thalamocortical terminals to investigate the role of this receptor in cortical development. Using quantitative in situ hybridization and receptor autoradiography, elevated levels of mRNA and binding-site expression were first detected at post-natal day 1 (P1) in deep and superficial layers, just beneath the AChE-stained thalamocortical terminals. Onset of expression occurred approximately 1 d after ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and binding-site expression exhibited a disjunctive, barrel-like pattern in layer IV and, more clearly, in layer VI. The mRNA and binding-site expressions peaked at approximately 1 week postnatal and then declined to adult levels. Unilateral electrolytic or cytochemical lesions placed in the thalamic ventrobasal complex at P0 (just as thalamocortical afferents are innervating the cortex) and at P6 (when the somatotopic map is well established) resulted in a marked reduction of alpha7 nAChR mRNA and [125]alpha-BTX binding-site levels in layers IV and VI, indicating their regulation by thalamocortical afferents. With P6 lesions, this reduction was observed as early as 6 hr postlesion. These results suggest that alpha7 nAChRs are localized primarily on cortical cells in rat somatosensory cortex and provide further evidence for thalamocortical influence on cortical ontogeny. These data also suggest a role for cholinergic systems during a critical period of cortical synaptogenesis.

Individual axon morphology and thalamocortical topography in developing rat somatosensory cortex.

The morphology of individual thalamocortical axons in developing rat primary somatosensory cortex was studied using lipophilic tracers. Anterograde labeling with lipophilic dyes demonstrated a topographical organization of thalamocortical projections exiting the thalamus as early as embryonic day (E) 16; retrograde labeling studies demonstrated topography of these projections as they reached the cortex as early as E18. At E17, axons course tangentially within the intermediate zone and turn or branch near the deepest layer of cortex (layer VIb), suggesting the presence of guidance cues in this region. Axons appear to grow and branch progressively within layers VIb and VIa during the following days; axons in the intermediate zone may give rise to radially directed branches. Individual axons appear to grow steadily and progressively into the cortex, with the leading front of axons at the transition zone between the cortical plate (CP) and the differentiating cortical layers. At birth (P0), thalamocortical axons extend radially through layers VIa and V and emit branches within these layers; some axons reach the CP. By P1, layer IV has begun to differentiate and axons begin to form a few simple branches in the vicinity of the layer IV cells. Over the ensuing week, axons generate more branches within layer IV, but the tangential extent of individual axon arbors does not exceed the width of a barrel. By P7, individual axons overlap within barrel clusters, and individual axons span the width of a cluster. These observations indicate that thalamic afferents develop by progressive growth of arbors that remain spatially restricted, rather than by overbranching and retracting arbors.