Development of the Limbic System

United States

Specific cortical and subcortical forebrain structures, often referred to as the limbic system, play a significant role in mediating emotional and motivated behavior as well as memory storage. The proper development of forebrain structures and the formation of neural circuitry in the forebrain are essential for the coordination and execution of adaptive behavior. Abnormal development of forebrain structures may underlie some neuropsychiatric disorders and increase vulnerability to addiction. Under this impetus and the revolution in molecular biology, a new focus on mechanisms specifying the formation of forebrain regions and synaptic connections in the forebrain has emerged. The speakers in this symposium describe some of the recent discoveries that have begun to elucidate the mechanisms underlying the development of limbic regions.

Abstracts

THE TRANSCRIPTION FACTOR NURR1 IS REQUIRED FOR DOPAMINE NEUROGENESIS. 

Lars Olson¹, Rolf Zetterstrom², Ludmila Solomin², Lottie Jansson², Barry Hoffer³, and Thomas Perlmann². ¹Department of Neuroscience, Karolinska Institute, Stockholm, ²The Ludwig Institute for Cancer Research, Stockholm Branch, ³Intramural Research Program, NIDA, NIH, Baltimore, MD.

Nurr1 belongs to a subfamily of orphan nuclear receptor transcription factors which also includes NGFI-B and Nor1. In situ hybridization demonstrates partly overlapping and partly unique cellular expression patterns in the developing and adult nervous system. These patterns will be reviewed. All three factors can bind to DNA as monomers and cause constitutive transcription. Interestingly, Nurr1 and NGFI-B, but not Nor1 can also heterodimerize with RXR. In such heterodimers, RXR can be activated by a vitamin A ligand. The functional and neuroanatomical studies therefore indicate possible redundancy between members of this subfamily of the steroid/thyroid hormone receptor superfamily. Midbrain dopamine (DA) neurons, however, express only Nurr1, making redundancy impossible in this region. Moreover, Nurr1 mRNA is found in the developing mesencephalic flexure area, prior to any known marker of DA neurons. When mice were generated in which functional Nurr1 mRNA expression had been eliminated, we found a complete absence of DA neurons. Detailed studies of Nurr1 -/- mice, using a host of markers for the DA neuron phenotype supports the conclusion that these neurons fail do be formed in the absence of the transcription factor. Nurr1 -/- mice die within 24 hours after birth, and appear unable to suckle and feed themselves. The exact mechanism behind this early postnatal lethality remains to be explained. Although the CNS and peripheral organs appear grossly normal in the knockouts, we currently favor the hypothesis that the animals succumb so soon after birth for other reasons than the lack of the nigrostriatal DA system.

MUTATIONS OF THE HOMEOBOX GENE DLX-1 AND DLX-2 DISRUPT THE STRIATAL SUBVENTRICULAR ZONE AND DIFFERENTIATION OF LATE-BORN STRIATAL NEURONS.

Stewart Anderson, Nina Ireland Laboratory of Developmental Neurobiology, University of California, San Francisco 94143-0984, USA.

The striatum plays a key role in normal brain function and in a number of disease processes, yet the molecular control of its development remains obscure. Recently, progress has been made due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they are important regulators of striatal development. Indeed, mice lacking both Dlx-1 and Dlx-2 have a time-dependent block in migration out of the striatal proliferative zones. Histological analysis of the Dlx-1/Dlx-2 mutant striatum on the day of birth (P0; also the day these mutants die) reveals an abnormal periventricular region of undifferentiated appearing cells, and a striatum-like region that is enriched for markers of the striatal patch compartment. Interestingly, the mutant periventricular region contains an abnormal overlap of dividing cells and cells immunoreactive for the post-mitotic neuronal marker, MAP2. This abnormality is first detectable at embryonic day 13.5 (E13.5). Two methods were used to determine that post-mitotic cells begin accumulating in the mutant striatal proliferative zone during the latter part of embryonic development. First, BrdU birthdating experiments revealed that cells born at E11.5 migrate into the differentiated portion of the mutant striatum by P0. However, most cells labeled at E15.5 remain within the mutant proliferative zone. Second, the study of migrating cells in slice cultures produced a similar result. The development of this migration abnormality in the Dlx-1/Dlx-2 mutants correlates with the emergence of the subventricular zone (SVZ) as the dominant proliferative population within the striatal anlage. In addition, the expression of several transcription factors is absent or abnormal in the mutant SVZ. These results suggest that differentiation within the striatal SVZ is disrupted in the mutants, and support the hypothesis that the striatal matrix compartment derives from the SVZ.

FUNCTIONAL MATURATION OF LIMBIC SYSTEM.

Harry T. Chugani, Departments of Pediatrics, Neurology and Radiology, Children's Hospital of Michigan, Wayne State University School of Medicine, Detroit, Michigan.

Using positron emission tomography (PET) and the tracer 2-deoxy-2(18F)fluoro-D-glucose (FDG), we have shown that the pattern of glucose metabolism in the human newborn brain is fairly consistent, with the highest degree of activity in primary sensory and motor cortex, cingulate cortex, medial temporal region, thalamus, brainstem and cerebellar vermis. Increases of glucose utilization are seen by two to three months in the parietal, temporal and primary visual cortex, basal ganglia, and cerebellar hemispheres. These changes in glucose metabolism coincide with improved skills involving visuo-spatial and visuo-sensorimotor integration, the disappearance or reorganization of brainstem reflex neonatal behaviors, and increasing cortical contribution to the electroencephalogram. Starting between 6 and 8 months, lateral and inferior portions of frontal cortex become more functionally active and eventually, between 8 and 12 months, the dorsal and med ial frontal regions also show increased glucose utilization. These changes of frontal cortex metabolism come at a time when cognitively-related behaviors, such as the phenomenon of stranger anxiety, and improved performance on the delayed response task begin to appear. Increased glucose requirement in frontal cortex also coincides with the expansion of dendritic fields and the increased capillary density observed in frontal cortex during the same period of development. By approximately one year of age, the infant's pattern of glucose utilization resembles qualitatively that of the adult.

At birth, the regional or local cerebral metabolic rates of glucose utilization (LCMRglc) are about 30% lower than those seen in adults. Between birth and approximately 3 years, the cerebral cortex shows a dramatic increase in LCMRglc to reach levels that exceed adult rates by over two-fold. Such changes in LCMRglc are not observed in brainstem, but a less dramatic increase is seen in basal ganglia and thalamus. Between 3 years and about 10 years, the LCMRglc for cerebral cortex is essentially at a high plateau of over two-fold the glucose utilization seen in adults. Subsequently, LCMRglc for cerebral cortex begins to decline and gradually reaches adult values by 16-18 years. Based on the temporal relationship between these developmental changes of LCMRglc and synaptogenesis in humans, as well as on similar studies performed in our laboratory on developing cat and monkey, we believe that the ontogenetic changes of LCMRglc described above provide an indirect measure of synaptogenesis in the human brain.

SPECIFICATION OF LIMBIC CORTICAL CIRCUITS.

Jurgen Bolz¹, Fanny Mann¹, Pat Levitt², ¹INSERM Unite’ 371 Cerveau et Vision, 18 ave du Doyen Lepine, 69500 Bron, France; ²University of Pittsburgh School of Medicine, Pittsburgh, USA.

One of the basic features distinguishing cortical areas is the pattern of specific afferent and efferent projection that define functional circuits. For example, during development thalamocortical afferents select their appropriate cortical region in a highly specific manner. This raises the possibility that there are regional specific cues with the cortex that guide thalamic afferents to select their cortical target area. The idea is supported by the expression patterns of the limbic system-associated membrane protein (LAMP) found primarily in the developing limbic areas of the cortex which was identified by Pat Levitt and colleagues. Transplantation studies of this group have show that the origin of cortical thalamic afferent innervating a cortical graft depends on its LAMP phenotype. To elucidate the role LAMP in the establishment of cortical connections, we analyzed the outgrowth of thalamic and cortical explants prepared from presumptive limbic and non-limbic regions on membrane substrates of either LAMP-expressing CHO cells or on membranes of non-transfected CHO cells (control). We found that length and sprouting of limbic thalamic and limbic cortical axons was enhanced on the LAMP substrate. Non-limbic thalamic fibers, however, responded by exhibiting reduce outgrowth compared to control. The length and branching behavior of neocortical fibers was not affected by LAMP. In a second set of experiments, explants were cultured on native postnatal membranes prepared from limbic or neocortical areas. Limbic thalamic and limbic cortical axons branched significantly more on membranes from limbic cortex, their target membranes, than on neocortical membranes. In contrast, non-limbic thalamic axons emitted more collaterals on membranes of neocortex. These branching preferences could be abolished by blocking LAMP on the membranes with a monoclonal antibody. Our results indicate that LAMP contributes to target recognition during cortical development through both attractive and repulsive mechanisms. Moreover, these experiments suggest the existence of membrane-bound molecules that specify neocortical areas as target for appropriate thalamic and cortical afferents.

REGULATION OF TOPOGRAPHIC PROJECTION IN THE LIMBIC SYSTEM BY THE EPH FAMILY MOLECULES.

Renping Zhou, Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08855.

Topographic projection is a general feature of brain architecture and is critical for appropriate information processing and coding. Nevertheless, little is known about the mechanisms that govern topographic organization. Among the many regions exhibiting topographic relations, the limbic system has been the focus of intense interest, since it plays key roles in learning, memory, and motivated behavior. The Eph family receptor tyrosine kinases and ligands have been recently implicated in the specification of topographic maps. We have shown that Eph family receptors and ligands are expressed in complementary fashion in neurons and targets, respectively, in several regions of the limbic system. For example, in the hippocampus, the Eph receptor Bsk is expressed in an increasing lateral to medial gradient. In contrast, at least three different ligands, Elf-1, LERK3/Ehk1-L, and AL-1/RAGS/LERK7, are transcribed in complementary (opposing) gradients in the hippocampal subcortical target, the lateral septum. However, the spatial and temporal distribution of the ligands are different such that combinatorially they form a smooth dosromedial to ventrolateral gradient in the lateral septum, specifying the full target region during development. Consistent with a key role in hippocamposeptal topographic projection, the ligands selectively inhibit the growth of the topographically inappropriate medial hippocampal neurites but sustain the growth of correct lateral neurites. Our studies indicate that the inhibitive interaction of Bsk and its ligands restrict the receptor-positive medial neurons to the topographically appropriate, ligand-poor dorsal septal target. In addition to the hippocamposeptal system, BSK and its ligands are also expressed in afferents and targets of neurons from several other regions of the limbic system, including neurons in the mesolimbic pathway, which arises from the ventral tegmental dopaminergic neurons and terminates in the nucleus accumbens and limbic regions such as the amygdala. These observations indicate that the Eph family molecules play important roles in establishing the limbic neural circuits.

MOLECULAR REGULATION OF LIMBIC CELL FATE AND CIRCUIT FORMATION

Pat Levitt and Kathie Eagleson, Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

The formation of unique areas of the cerebral cortex during development is prerequisite for functional specialization. We have extended our earlier transplant studies to show that progenitor cells are particularly sensitive to environmental signals that control areal fate determination. Using expression of the limbic system-associated membrane protein (LAMP) as an assay for limbic phenotype, we have shown that progenitors isolated from different domains of presumptive neocortex and grown in culture can respond to LAMP-inducing signals. Members of the epidermal growth factor receptor family, including those responsive to TGF and heregulin, can mediate cell fate decisions that occur independent of effects on proliferation or survival. These early decisions by progenitors to express a limbic molecular phenotype have implications for subsequent events of cortico-cortical and thalamo-cortical connectivity that underlie normal limbic system function.