Neuronal plasticity in primate telencephalon: anomalous projections induced by prenatal removal of frontal cortex.

When the dorsolateral prefrontal cortex in one hemisphere of a rhesus monkey is resected 6 weeks before birth and the fetus survives to postnatal ages, neurons of the corresponding cortex in the intact hemisphere issue a greatly expanded projection to the contralateral caudate nucleus in addition to a normal projection to the ipsilateral caudate. The enhancement of the crossed prefronto-caudate pathway after prenatal neurosurgery provides direct evidence for lesion-induced neuronal rearrangement in the primate telencephalon.

Delayed recovery of function following orbital prefrontal lesions in infant monkeys.

Monkeys given orbital prefrontal lesions at 1, 4, or 8 weeks of age exhibited a severe learning disability when they were tested at 1 year of age, but showed substantial recovery by the time they were 2 years old. These results suggest that the protracted maturation of intact cortical regions is important in recovery of function after early brain injury.

Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey.

Depletion of dopamine in a circumscribed area of association cortex in rhesus monkeys produces an impairment in spatial delayed alternation performance nearly as severe as that caused by surgical ablation of the same area. This behavioral deficit can be pharmacologically reversed with dopamine agonists such as L-dopa and apomorphine. These data provide direct evidence that dopamine plays an important role in a specific cortical function.

Sex-dependent behavioral effects of cerebral cortical lesions in the developing rhesus monkey.

Male rhesus monkeys with orbital prefrontal lesions were imtpaired on behavioral tests at 2(1/2) months of age whereas similar deficits were not detected in females with comparable lesions until 15 to 18 months of age. The results suggest that the maturation of a cortical region in the primate brain proceeds at different tempos in males and females.

An intricately patterned prefronto-caudate projection in the rhesus monkey.

The distribution of prefronto-caudate fibers in the caudate nucleus was studied autoradiographically in monkeys of various ages in which tritiated amino acids had been injected into the middle one-third of the length of the dorsal bank of the principal sulcus. The results indicate that, contrary to previous reports which had suggested a projection to only the head of the caudate nucleus, area 9 of Brodmann projects to the entire length of the nucleus. In the head of the caudate nucleus the cortico-caudate fibers are distributed in a pattern which is remarkable in two respects. First, the grains are not uniformly distributed but rather are segregated into clusters separated from one another by territories in which grain density does not exceed background. Second, individual clusters of grains, circular or elliptical in shape, surround grain free cores. These patterns of fiber distribution within the head of the nucleus are more sharply defined in newborn than in older monkeys. Our findings suggest that the caudate nucleus is organized more as an anatomic and functional mosaic than as the homogeneously organized structure that it is commonly considered to be.

Postnatal maturation of subcortical projections from the prefrontal cortex in the rhesus monkey.

Orbital and dorsolateral prefrontal lesions were performed on a series of rhesus monkeys at 2, 6, or 24 months of age. The consequent degeneration in the efferent pathways from these cortical regions to the caudate nucleus, the dorsomedial nucleus of the thalamus and adjacent structures was studied at 5- and 15-day survival times by a modification of the Nauta-Gygax method for tracing degenerating fibers. Following dorsolateral lesions, considerable numbers of black-impregnated degenerating fibers were found in the parvocellular division of the dorsomedial nucleus and in the fiber bundles of the internal capsule and the subcallosal fasciculus at all ages.

Prenatal removal of frontal association cortex in the fetal rhesus monkey: anatomical and functional consequences in postnatal life.

Anatomical and behavioral consequences were examined following bilateral or unilateral resections of the presumptive dorsolateral prefrontal cortex in 4 rhesus monkey fetuses at E102 (102nd embryonic day), E104, E106 and E119, with subsequent replacement in utero and delivery at later stages of development. Unoperated monkeys of appropriate pre- and postnatal ages served as controls for certain features of morphogenesis and tectogenesis. Monkeys given comparable unilateral or bilateral lesions at selected postnatal ages were used as controls for the behavioral effects of prenatal resection as well as for neuropathological examination. The behavioral evaluation provided evidence for extraordinary preservation of function after frontal cortical resection in the prenatal period. During the entire course of its postnatal development, a monkey that sustained bilateral resection as a fetus at E106 performed surprisingly well and clearly with the competence of unoperated monkeys of identical or older ages on tasks selective for dorsolateral prefrontal cortical function; it did not exhibit deficits characteristic of animals that have comparable or even smaller resections as juveniles or adults. Following either unilateral or bilateral resection prior to E106, ectopic sulci and gyri appeared in intact regions of the frontal lobe of each hemisphere, as well as in locations at a considerable distance from the lesions, such as the temporal and occipital lobes. The cytoarchitectonic characteristics of the cortex in these anomalous gyri were typical of adjacent cortex in the regions in which they appeared. Examination of the parvocellular subdivision of the mediodorsal thalamic nucleus yielded the novel finding that this principal source of essential projections to the dorsolateral prefrontal cortex contained a virtually normal number of neurons in postnatal monkeys, following unilateral or bilateral lesions performed at or before E106, and exhibited only a partial loss of neurons after surgery at E119. The same cells regularly degenerate following comparable resections in the postnatal period. The survival of these neurons in prenatally operated monkeys is unprecedented, and can be explained by several hypotheses, including the possibility that their axons become rerouted to their structures in the absence of their normal targets. The present results thus demonstrate that ablation of a circumscript area of the frontal cortex in a non-human primate a full two months before birth may leave the functions later to be subserved by that area unaffected. Such early surgical intervention results in significant alteration in the gross morphological characteristics of the cortex, and apparently induces a functionally compensatory reorganization of thalamocortical connections. Similar principles may account for sparing of function following early damage to the human brain.

Functional development of the dorsolateral prefrontal cortex: an analysis utlizing reversible cryogenic depression.

The dorsolateral prefrontal cortex of rhesus monkeys was functionally inactivated by local hypothermia as the monkeys performed spatial delayed-response and spatial delayed-alternation tasks at different stages of postnatal development. Cryogenic depression of prefrontal cortex at a temperature sufficient to induce 21--25% decrements in delayed-response performance in 34--36-month-old-monkeys, produced deficits of only 7--8% in 19--31-month-old and no detectable loss in younger monkeys, 9--16 months of age. Delayed-alternation performance was impaired by local hypothermia as early as 8.5 months of age, but maximal cooling-induced deficits on this task were not observed before 33 months of age. Thermal gradients mapped in representative monkeys at different stages of development were remarkable similar, indicating that the age-dependent differences in behavior were not attributable to technical factors. The results obtained in the present study on normal developing monkeys confirm the interpretation of previous research on brain-damaged infants that functional maturation of the dorsolateral prefrontal cortex is protracted over several years of postnatal life, and extends the earlier studies by indicating that the lower limit for maturity of dorsolateral function is close to puberty in this species. Further, the present study revealed that delayed-response and delayed-alternation performance are dissociable dorsolateral functions which achieve maturity at different rates. The convergence of evidence from reversible neural depression and permanent lesion methods provides strong validation for neurobehavioral analysis as a general approach to the study of regional maturation of the brain.

Columnar distribution of cortico-cortical fibers in the frontal association, limbic, and motor cortex of the developing rhesus monkey.

The terminal distribution of cortico-cortical connections was examined by autoradiography 7-8 days following injections of tritium labeled amino acids into the dorsal bank of the principal sulcus, the posterior part of the medial orbital gyrus, or the hand and arm area of the primary motor cortex in monkeys ranging in age from 4 days to 5.5 months. Labeled axons originating in these various regions of the frontal lobe have topographically diverse ipsilateral and contralateral destinations but virtually all of these projections share a common mode of distribution: they terminate in distinct vertically oriented columns, 200-500 mum wide, that extend across all layers of cortex and alternate in regular sequence with columns of comparable width in which grains do not exceed background. Spatial periodicity in the pattern of transported label in such regions as the prefrontal association cortex, the retrosplenial limbic cortex and the motor cortex indicates that columination in the intracortical distribution of afferent fibers is not unique to sensory specific cortex but is instead a general feature of neocortical organization. A columnar mode of distribution of cortico-cortical projections is present in monkeys at all ages investigated but is especially well delineated in the youngest of them. Thus, grain concentrations within columns are very high in monkeys injected at 4 days of age, somewhat lower in monkeys injected at 39-45 days of age, and least dense in those injected at 5.5 months. The distinctness of the spatially segregated pattern of innervation in the cortex of neonates indicates that the columnar organization of association-fiber systems in the frontal and limbic cortex is achieved before or shortly after birth.

Regional distribution of monoamines in the cerebral cortex and subcortical structures of the rhesus monkey: concentrations and in vivo synthesis rates.

Endogenous monoamine concentrations and turnover rates vary markedly in different regions of neocortex as well as in various subcortical structures of young adult rhesus monkeys. Monoamine levels and synthesis rates in amygdala, hippocampus, neostriatum, thalamus and brain stem are generally similar to comparable measures previously reported in a variety of species. However, extending and confirming the results of an earlier study, cortical monoamines exhibit topographically specific patterns of distribution. Thus, dopamine concentration is highest in the prefrontal and temporal neocortex; it decreases along the fronto-occipital axis and only trace amounts are detectable in the visual cortex. The distribution of norepinephrine is similar to that of dopamine except that the highest concentrations of norepinephrine are found in somatosensory cortex instead of prefrontal cortex. The pattern of distribution of serotonin is more uniform. However, the distribution of its metabolite, 5-hydroxyindoleacetic acid, is complementary to that of dopamine: the concentration is lowest in prefrontal cortex and highest in posterior regions of the telencephalon. Synthesis of catecholamines as measured by DOPA accumulation in monkeys treated with an aromatic amino acid decarboxylase inhibitor, NSD 1015, generally parallels the distribution of the catecholamines while indoleamine synthesis, as measured by 5-HTP accumulation, is similar to the distribution of 5-hydroxyindoleacetic acid. It may be significant that synthesis rates for the catecholamines are especially high in various areas of association cortex.

Identification of four brain areas each enriched in a unique muscarinic receptor subtype.

The affinities of muscarinic agonists and antagonists were determined by autoradiography and image analysis in selected areas of the rat brain. IC50 values and Hill coefficients for the inhibition of the binding of 0.2 nM [3H]-QNB to dentate gyrus, superior colliculus, rhomboid thalamus and substantia nigra were measured in coronal sections. Pirenzepine displayed a high affinity for receptors in the dentate gyrus and AF-DX 116, the superior colliculus. Both pirenzepine and AF-DX 116 had high affinities for the substantia nigra and low affinities for the rhomboid thalamus. Gallamine displayed a 50-fold preference for superior colliculus over dentate gyrus receptors. Amitriptyline was less selective, showing a modest preference for substantia nigra receptors and 4-DAMP was essentially nonselective. Carbachol was the most selective agonist with a 4000-fold preference for superior colliculus over dentate gyrus receptors. Other agonists except RS 86 were also selective for superior colliculus receptors in the order carbachol much greater than arecoline greater than bethanechol greater than McN A343 = oxotremorine = pilocarpine.

Regulation of expression and function of muscarinic receptors.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Molecular analysis of the regulation of muscarinic receptor expression and function.

Several systems are being used to determine the molecular and cellular basis for the regulation of expression and function of the muscarinic receptors. Treatment of chick heart cells in culture results in decreased levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription which requires concomitant mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. Site-directed mutagenesis was used to demonstrate that the single tyrosine residue in the carboxyl-terminal cytoplasmic tail of the m2 receptor is involved in agonist-induced down-regulation but not sequestration. Activation of heterologous receptors in chick heart cells can also regulate mAChR mRNA levels. A cAMP-regulated luciferase reporter gene, has been used to demonstrate that the m4 receptor preferentially couples to Gi alpha-2 or Go alpha over Gi alpha-1 or Gi alpha-3 to mediate inhibition of adenylyl cyclase activity. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we are beginning to use the method of targeted gene disruption by homologous recombination to generate mice deficient in specific receptor subtypes.

Developmental determinants of cortical plasticity.

A central problem in neuropsychology concerns the mechanisms by which functions are restored following injury to the brain. Functional recovery is particularly striking following damage to the cerebrum in early life and accordingly our studies deal with the effects of prefrontal cortical lesions performed on infant monkeys. We describe here two essentially different patterns of behavioral recovery following selective lesions of the dorsolateral and orbital prefrontal cortex. Following orbital lesions in infancy, the monkeys are impaired when tested initially between 12 and 18 months of age but give evidence of substantial recovery when they are retested at 24 months of age. In contrast, monkeys given dorsolateral resection early in life exhibit sparing of function during the initial period of testing but this impunity usually associated with dorsolateral injury in infancy gives way later to a picture of retarded development. We attribute these two patterns of recovery - progressive and regressive - to differential rates of development of the two prefrontal cortical regions involved. Our evidence suggests that while orbital cortex becomes functionally mature by 1 year of age and probably earlier, the elaboration of dorsolateral function is delayed well into the second year of life. We propose that the dorsolateral cortex, by virtue of its relative immaturity at the time of orbital excision, can come in time to subsume the functions of the orbital cortex but that the orbital cortex may be functionally "committed" too early in life to compensate reciprocally for injury sustained by the dorsolateral cortex.