Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones.

Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton. While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development. Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth. Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted. The opposite phenotype was observed when a constitutively active RhoA variant was expressed. Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3(lim) or Staphylococcus aureus, C3(stau2)), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3(bot)) or enzymatically dead C3(bot) significantly increased axon growth and axon branching. Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation. However, extracellular application of enzymatically active or dead C3(bot) exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.

Lateral magnocellular nucleus of the anterior neostriatum (LMAN) in the zebra finch: neuronal connectivity and the emergence of sex differences in cell morphology.

The song system of birds provides a model system to study basic mechanisms of neuronal plasticity and development underlying learned behavior. Song learning and production involve discrete sets of interconnected nuclei in the avian brain. One of these nuclei, the lateral magnocellular nucleus of the anterior neostriatum (LMAN), is the output of the so-called anterior forebrain pathway known to be essential for learning and maintenance of song, both processes depending on auditory feedback. In zebra finches, only males sing and this sexually dimorphic behavior is mirrored by sexual dimorphism in neuronal structure that develops during ontogeny. Female zebra finches are not able to sing and nuclei of the song system are strongly reduced in size or even lacking, when compared to male brains. Only LMAN can be delineated as easily in females as in males. Since female zebra finches, despite being unable to sing, recognize song just as males do and form a memory for song (model acquisition) early in life, LMAN is a putative candidate for song acquisition in both sexes. Therefore, development of LMAN was studied at the cellular and ultrastructural level in both male and female zebra finches. Regressive development of dendritic spines, enlargement of neuronal cell body and nuclei size, as well as changes at the nucleolar level are events all occurring exclusively in males, when song learning progresses. The decline in synapse number and the augmentation in synaptic contact length at synapses in LMAN in males are indicative for synaptic plasticity, whereas in females synapse number and synaptic contact length remain unchanged.

Enlargement of neuronal somata in the LMAN coincides with the onset of sensorimotor learning for song.

The lateral magnocellular nucleus of the anterior neostriatum (LMAN) in the zebra finch (Taeniopygia guttata) has been shown to play a developmentally restricted role that is essential during song-learning processes. Dendritic spine frequencies and synapse numbers in LMAN have been reported to decline in males during early vocal motor learning and thereafter, but not in females, who do not sing. Nissl staining has shown the LMAN volume to be very similar in both sexes, however. To gain more insight into the development of sex-specific differences in LMAN, the size of neuronal somata and cell nuclei were analyzed in micrometer semithin sections. Cell somata and nuclei were similar in males and females during the initial phases of sensory memory formation for song, but during early vocal motor learning cell size increased in males and decreased in females. Sex differences in neuronal somata size were present at 50 days and remained throughout life. This sex difference may be indicative of a difference in protein biosynthesis in LMAN, arising as a consequence of vocal learning in males.

Nucleolar size and shape is sexually dimorphic in neurons of the lateral magnocellular nucleus of the anterior neostriatum (LMAN) in birds.

The lateral magnocellular nucleus of the anterior neostriatum (LMAN) in birds plays an important role in songlearning processes. Recently, it has been shown that structural changes at the cellular level in males are causally related to vocal learning. Whereas males sing, females do not. This sexual difference in behavior is also reflected in sexual differences in the neuronal structure in adult birds, with males having larger neuronal somata than do females. In the present report, the size and shape of the nucleoli were investigated to determine if sexual differences were also present at the nucleolar level. The data demonstrated a strong sexual difference in nucleolar size in both juvenile and adult birds, the cross-sectional area of the nucleoli being significantly larger in males than in females (30% and 50% larger in juvenile and adult birds, respectively). This difference between males and female finches was also reflected by the cross-sectional area of a specific type of nucleolus exhibiting a central light area. In both sexes, nucleoli exhibiting a central light area were significantly larger in juvenile and adult birds than nucleoli that lacked a central light area. The percentage of neurons exhibiting a central light area was higher in adult males than in adult females, but not in juvenile birds. The time course of development of nucleoli exhibiting a central light area in males was very similar to the development of neuronal somata size in LMAN neurons. The larger size of the nucleoli in LMAN neurons in males and the developmental changes in the incidence of nucleoli exhibiting a central light area may be indicative of a high level of ribosome production necessary for song-learning processes to occur.

Divergent and parallel development in volume sizes of telencephalic song nuclei in male and female zebra finches.

Song control regions in passerine birds are known to be sexually dimorphic in the adult brain in species like zebra finches in which most males sing whereas females do not. As a first step toward the analysis of the establishment of anatomical sex differences, volumetric changes of Nissl-defined song control regions in the forebrain of the zebra finch have been quantified in both sexes at 10-day intervals starting at day 10 posthatching. In males, the Nissl-defined volume of the high vocal center, the robust nucleus of the archistriatum, and area X of the lobus parolfactorius increased with age, reaching the adult value at 60, 50, and at 40 days posthatching, respectively. The lateral magnocellular nucleus of the anterior neostriatum increased in volume until 20 days and then gradually decreased to reach the adult value at about 40 days. Whereas area X is absent in females, the high vocal center, the robust nucleus of the archistriatum, and the lateral magnocellular nucleus of the anterior neostriatum were detectable throughout development and in adulthood. In contrast to the males, volumes of the high vocal center and of the robust nucleus of the archistriatum decreased in females between 10 and 40 days posthatching (58% and 61%, respectively), when adult values were reached. Contrary to the development of these two nuclei in females, the volumetric development of the lateral magnocellular nucleus of the anterior neostriatum was very similar in both sexes. Females began with a smaller lateral magnocellular nucleus of the anterior neostriatum at 10 days posthatch, which led to a sexual dimorphism in juvenile stages, but there was no sexual dimorphism of volume size in adult brains.

Electrophysiological and morphological evidence for a new projection of LMAN-neurones towards area X.

Neuronal connectivity of brain areas involved in song learning and song production was studied in the in vitro slice preparation of the zebra finch brain by electrophysiological intracellular recording techniques and by micro-injections of the fluorescent tracer tetramethyl-rhodamine-dextran-amine. While validating some of the known projections to be preserved in the in vitro slice preparation, we were also able to identify a new projection from neurones of the lateral portion of the magnocellular nucleus of the anterior neostriatum towards area X of the lobus parolfactorius.

Song isolation is associated with maintaining high spine frequencies on zebra finch 1MAN neurons.

Male zebra finches normally learn much of their song during the second month after hatching. This is a period of rapid change throughout the brain. We studied anatomical consequences of manipulating exposure to song. We investigated neurons of lateral MAN (1MAN), a nucleus implicated in song learning (Bottjer et al., 1984), in male and female zebra finches (Taeniopygia guttata) at 55 days posthatch. Birds were raised either under normal colony conditions (social) or in colonies in which adult males were removed when the young hatched (song deprived). Brains were stained with the Golgi-Cox method. Fine morphological details of spiny 1MAN neurons were recorded with a 3D semiautomated computer system. Several features of the spiny 1MAN neurons differ between sexes. Males have neurons with larger somata, more primary dendrites and thicker dendrites, than neurons from females. These features as well as dendritic length and other branching characteristics do not differ between treatment groups. There is a large difference in dendritic spine frequencies between the social and the song-deprived groups. Social, song-experienced males have spine frequencies 41% lower than song-deprived males. In females, spine frequencies are as high as in the song-deprived males and do not differ between the song-deprived and social conditions. Developmental overproduction and subsequent pruning of neural connections have been observed in many areas of the central nervous system. We suggest that apparent pruning in 1MAN is modulated by experience: It takes place if the social experiences associated with auditory song learning have occurred. This finding is consistent with the synapse selection hypothesis of Changeux and Danchin Brain Research, 309, (1976) and with data found using an acoustic filial imprinting paradigm in the domestic chicken.

Regressive development in neuronal structure during song learning in birds.

We investigated the development of spiny neurons in the lateral magnocellular nucleus of the anterior neostriatum before, during, and after song learning in male zebra finches (Taeniopygia guttata). The frequency of dendritic spines, dendritic field size, and branching characteristics were quantified at different ages in Golgi-stained tissue using a three-dimensional computerized tracing system. During development, overall spine frequencies increase between 3 and 5 weeks and decrease thereafter. In particular, spine frequencies of middle segments decrease significantly by 14% between 5 and 7 weeks posthatching (p = 0.017). A further reduction of 48% occurs between 7 weeks and adulthood (p < 0.001), resulting in a spine reduction of 56% on middle segments between 35 days of age and adulthood. In addition to the reduction of spine frequencies, we find regressive events also on some of the neuronal parameters that we have quantified. In general, dendrites of adult animals terminate closer to the cell body than those of 7-, 5-, or 3-week-old birds. Whereas no changes in segment length of first- and second-order dendrites have been identified, third-order dendrites end 19% closer to the cell body in adults than in younger birds (p < 0.024). Second-order dendrites in adult animals branch less frequently than in 3-week-old animals (35%, p = 0.017). There is also a trend of a smaller number of tertiary branches in adulthood compared with 3-week-old birds (41%, p = 0.060). The morphological changes may be related to the function of this nucleus and the sensitive phase for song acquisition.

Developmental changes in the number, size, and orientation of GFAP-positive cells in the CA1 region of rat hippocampus.

Changes in extracellular potassium concentration as measured with ion-selective microelectrodes revealed abnormally large accumulations in the hippocampus during postnatal development. While rises in [K+]o during stimulation of the Schaffer collaterals were limited to about 12 mM in adult animals, identical stimulations elicited rises to levels as large as 18 mM in juveniles. Since astrocytes are believed to play an important role in K+ homeostasis, we studied the postnatal development of astrocytes in the CA1 region of rat hippocampus in four age groups using a polyclonal antibody against glial fibrillary acidic protein (GFAP). The main proliferation of GFAP-positive cells (GFAPpc) occurred in all laminae between postnatal days 8 and 16. The number of GFAP-positive astrocytes per unit area was reached in stratum lacunosum-moleculare and stratum oriens at about 2 weeks and in stratum radiatum at about 3 weeks of age. During further development--at the age of 24 days--the orientation of individual astrocytes in stratum radiatum became polar with an orientation almost perpendicular to stratum pyramidale. This was revealed by an analysis based on determination of the quotients between the angular orientation of the processes of single individual GFAP-positive cells. When the crossing points of all glial processes over vertical and horizontal grid lines were determined and respective quotients evaluated, the same development towards a perpendicular orientation of astrocytes was noted in stratum radiatum. The same approach revealed a transient orientation parallel to the fissure in stratum lacunosum-moleculare around day 24. Camera lucida drawings of GFAPpc in stratum radiatum revealed that astrocytes became larger during the first three postnatal weeks, followed by a reduction of various parameters (e.g., cell extension, branching pattern) until adulthood. The observed developmental changes of astroglial cells may contribute to the known delayed maturation of potassium regulation in rat hippocampus.