Bird song represents a powerful model system for many of the important problems in behavioral neurobiology, offering both easily measured sensory and motor patterns and a discrete neural effector system. Methods were developed to record the discharge of neurons in singing birds to examine the functions of nuclei in the song control pathway previously implicated anatomically. In several cases, lesions and other techniques were employed to test predictions derived from electrode recordings. Four major findings emerge from these studies. Single-unit recordings from telencephalic nucleus hyperstriatum ventrale, pars caudale (HVc) show several classes of neurons with apparently specialized roles in song production and/or sensorimotor interaction. The nucleus interfacialis (Nlf; Nottebohm, 1980), which provides an input to HVc and is anatomically the "highest" nucleus in the descending motor pathway, is uniquely placed among vocal control nuclei to be a generator of timing cues for song. Consistent with the unidirectional connections between nuclei of the descending pathway, Nlf, HVc, and nucleus robustus archistriatalis (RA) are activated sequentially prior to sound onset. Three other nuclei with connections to or from the descending tract do not show song-related activity in the adult. Bilateral HVc recordings and peripheral disruptions of the vocal apparatus suggest that both hemispheres and syringeal halves normally make similar contributions to most if not all song syllables. The latter finding casts doubt on the analogy between neural lateralization in bird song and in human speech.

Discrete telencephalic nuclei HVc (hyperstriatum ventrale, pars caudale) and RA (nucleus robustus archistriatalis) have been implicated by lesion studies in the control of vocalization in songbirds. We demonstrate directly the role of HVc in vocalization by presenting neuronal recordings taken from HVc of singing birds. Intracellular recordings from anesthetized birds have shown that many neurons in HVc respond to auditory stimuli. We confirm this result in the extracellular recordings from awakebehaving birds and further demonstrate responses ofHVc neurons to playback of the bird's own song. The functional significance of these reponses is not yet clear, but behavioral studies show that auditory feedback plays a crucial role in the development of normal song. We show that the song-correlated temporal pattern of neural activity persists even in the deaf bird. Furthermore, we show that in the normal bird, the activity pattern correlated with production of certain song elements can be clearly distinguished from the pattern of auditory responses to the same song elements. This result implies that an interaction occurs in HVc ofthe singing bird between motor and auditory activity. Through experiments involving playback of sound while the bird is singing, we show that the interaction consists of motor inhibition of auditory activity in HVc and that this inhibition decays slowly over a period ofseconds after the song terminates.The discovery of a set ofdiscrete brain nuclei implicated in the control of vocalization in songbirds (1) was an important step toward understanding the neural basis for this complex behavior. A series of lesion studies by Nottebohm and colleagues demonstrated severe song deficits after damage to either of the forebrain nuclei HVc (hyperstriatum ventrale, pars caudale) or RA (nucleus robustus archistriatalis). Behavioral studies (2-4) have shown that the timing and spectral characteristics of song elements are learned by males from adult birds and that this learning process occurs in two phases that can be temporally distinct: an auditory phase in which a model of the tutor song is stored in the brain and a motor phase in which the bird's own vocalizations are progressively matched to the song model. A bird deafened before the onset of singing cannot vocally reproduce the stored song model (5), suggesting that there should be a regulatory connection between the auditory and the vocal motor control systems.Katz and Gurney (6) have recently demonstrated, using an intracellular recording technique, that many neurons in HVc ofthe zebra finch (Poephila guttata) respond to auditory stimuli. They also observed auditory responses in cells ofthe neostriatal shelf area underlying HVc, which receives afferents from the avian forebrain auditory area known as field L (7). Although the significance and specificity ofauditory responses in HVc are not yet clear, the fact that auditory information is available to the motor system controlling song production carries an obvious suggestion of involvement ...

There is strong evidence that growthassociated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (؊͞؊) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to ؊͞؊ cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whiskerspecific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 ؊͞؊ mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex.

Focal injections of horseradish peroxidase (HRP) in dimethylsulfoxide (DMSO) were targeted into mouse somatosensory cortex, in vitro, with a template. Injections were made at different depths and in different locations in the whisker-barrel-defined somatosensory map in order to determine quantitative connectivity patterns within and between barrel-defined cortical columns. Cortices were sectioned in a plane parallel to the pia at 75 microns. Data were collected directly from microscope slides by computer. Data are presented as: 1) Plots of computer-mapped HRP reaction product density in neurons and cell locations for each section in relation to barrel boundaries; 2) histograms of label in cortical layers related to individual barrel-defined columns; 3) polar plots of relative amounts of label within individual barrel columns in sections through each barrel column; 4) vectors which represent HRP reaction product density as a function of direction and distance from the injection site; 5) statistical analysis of the shape of the label distribution pattern in the plane of the cortex as a function of injection site depth; and 6) probability of labeling of any other barrel column given a labeled barrel column. The principal findings are: 1) The pattern of label distribution, after an injection directly above or directly below an individual barrel, is hour-glass shaped with the waist of the hour-glass in layer IV. 2) Connections within barrel cortex are asymmetrical. Barrel-related columns within a row are more strongly interconnected than those in different rows. 3) Connections of the small barrels associated with whiskers on the upper lip are strongest with other small barrels, but strong connections also exist between these small barrels and the larger barrels. 4) The pattern of intracortical connections in SII is not asymmetrical; interlaminar connections in SII are fundamentally different from those in barrel cortex. 5) Quantitative intracortical projection patterns are highly consistent with functional data on intracortical processing of whisker information. As such, the quantitative data clearly indicate the spatial extent and relative magnitude of populations of neurons involved in intracortical processing of sensory information. The spatial arrangements of these intracortical connections, in conjunction with known developmental events, make it highly likely that the distribution of intracortical axons in mouse barrel cortex is sculpted in part by experience.

Cortical columns associated with barrels in layer IV of the somatosensory cortex were characterized by high-resolution 2-deoxy-D-glucose (2DG) autoradiography in freely behaving mice. The method demonstrates a more exact match between columnar labeling and cytoarchitectonic barrel boundaries than previously reported. The pattern of cortical activation seen with stimulation of a single whisker (third whisker in the middle row of large hairs--C3) was compared with the patterns from two control conditions--normal animals with all whiskers present ("positive control")--and with all large whiskers clipped ("negative control"). Two types of measurements were made from 2DG autoradiograms of tangential cortical sections: 1) labeled cells were identified by eye and tabulated with a computer, and 2) grain densities were obtained automatically with a computer-controlled microscope and image processor. We studied the fine-grained patterns of 2DG labeling in a nine-barrel grid with the C3 barrel in the center. From the analysis we draw five major conclusions. 1. Approximately 30-40% of the total number of neurons in the C3 barrel column are activated when only the C3 whisker is stimulated. This is about twice the number of neurons labeled in the C3 column when all whiskers are stimulated and about ten times the number of neurons labeled when all large whiskers are clipped. 2. There is evidence for a vertical functional organization within a barrel-related whisker column which has smaller dimensions in the tangential direction than a barrel. There are densely labeled patches within a barrel which are unique to an individual cortex. The same patchy pattern is found in the appropriate regions of sections above and below the barrels through the full thickness of the cortex. This functional arrangement could be considered to be a "minicolumn" or more likely a group of "minicolumns" (Mountcastle: In G.M. Edelman and U.B. Mountcastle (eds): The Material Brain: Cortical Organization and the Group-Selective Theory of Higher Brain Function. Cambridge: MIT Press, '78). 3. Within the stereotyped geometry of the barrel field, there is considerable individual variation in the radial labeling distribution in corresponding (homotypical) columns of different cerebral hemispheres. This result is consistent with the hypothesis that dynamic processes operate to determine the connection strengths between neural elements in somatosensory cortex. It provides a basis for testing various "connectionist" and "group selection" theories of neural organization and development.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic (5-HT) axons from the raphe nuclei are among the earliest afferents to innervate the developing forebrain. The present study examined whether GAP-43, a growth-associated protein expressed on growing 5-HT axons, is necessary for normal 5-HT axonal outgrowth and terminal arborization during the perinatal period. We found a nearly complete failure of 5-HT immunoreactive axons to innervate the cortex and hippocampus in GAP-43-null (GAP43-/-) mice. Abnormal ingrowth of 5-HT axons was apparent on postnatal day 0 (P0); quantitative analysis of P7 brains revealed significant reductions in the density of 5-HT axons in the cortex and hippocampus of GAP43-/- mice relative to wild-type (WT) controls. In contrast, 5-HT axon density was normal in the striatum, septum, and amygdala and dramatically higher than normal in the thalamus of GAP43-/- mice. Concentrations of serotonin and its metabolite, 5-hydroxyindolacetic acid, and norepinephrine were decreased markedly in the anterior and posterior cerebrum but increased in the brainstem of GAP43-/- mice. Cell loss could not account for these abnormalities, because unbiased stereological analysis showed no significant difference in the number of 5-HT dorsal raphe neurons in P7 GAP43-/- versus WT mice. The aberrant 5-HT innervation pattern persisted at P21, indicating a long-term alteration of 5-HT projections to forebrain in the absence of GAP-43. In heterozygotes, the density and morphology of 5-HT axons was intermediate between WT and homozygous GAP43-/- mice. These results suggest that GAP-43 is a key regulator in normal pathfinding and arborization of 5-HT axons during early brain development.

There is an urgent need for animal models of autism spectrum disorder (ASD) to understand the underlying pathology and facilitate development and testing of new treatments. The synaptic growth-associated protein-43 (GAP43) has recently been identified as an autism candidate gene of interest. Our previous studies show many brain abnormalities in mice lacking one allele for GAP43 [GAP43 (+/−)] that are consistent with the disordered connectivity theory of ASD. Thus, we hypothesized that GAP43 (+/−) mice would show at least some autistic-like behaviors. We found that GAP43 (+/−) mice, relative to wild-type (+/+) littermates, displayed resistance to change, consistent with one of the diagnostic criteria for ASD. GAP43 (+/−) mice also displayed stress-induced behavioral withdrawal and anxiety, as seen in many autistic individuals. In addition, both GAP43 (+/−) mice and (+/+) littermates showed low social approach and lack of preference for social novelty, consistent with another diagnostic criterion for ASD. This low sociability is likely because of the mixed C57BL/6J 129S3/SvImJ background. We conclude that GAP43 deficiency leads to the development of a subset of autistic-like behaviors. As these behaviors occur in a mouse that displays disordered connectivity, we propose that future anatomical and functional studies in this mouse may help uncover underlying mechanisms for these specific behaviors. Strain-specific low sociability may be advantageous in these studies, creating a more autistic-like environment for study of the GAP43-mediated deficits of resistance to change and vulnerability to stress.

Electrophysiological data from the rodent whisker/barrel cortex indicate that GABAergic, presumed inhibitory, neurons respond more vigorously to stimulation than glutamatergic, presumed excitatory, cells. However, these data represent very small neuronal samples in restrained, anesthetized, or narcotized animals or in cortical slices. Histochemical data from primate visual cortex, stained for the mitochondrial enzyme cytochrome oxidase (CO) and for GABA, show that GABAergic neurons are more highly reactive for CO than glutamatergic cells, indicating that inhibitory neurons are chronically more active than excitatory neurons but leaving doubt about the short-term stimulus dependence of this activation. Taken together, these results suggest that highly active inhibitory neurons powerfully influence relatively inactive excitatory cells but do not demonstrate directly the relative activities of excitatory and inhibitory neurons in the cortex during normal behavior.We used a novel double-labeling technique to approach the issue of excitatory and inhibitory neuronal activation during behavior. Our technique combines high-resolution 2-deoxyglucose (2DG), immunohistochemical staining for neurotransmitterspecific antibodies, and automated image analysis to collect the data. We find that putative inhibitory neurons in barrel cortex of behaving animals are, on average, much more heavily 2DG-labeled than presumed excitatory cells, a pattern not seen in animals anesthetized at the time of 2DG injection. This metabolic activation is dependent specifically on sensory inputs from the whiskers, because acute trimming of most whiskers greatly reduces 2DG labeling in both cell classes in columns corresponding to trimmed whiskers. Our results provide confirmation of the active GABAergic cell hypothesis suggested by CO and single-unit data. We conclude that strong activation of inhibitory cortical neurons must confer selective advantages that compensate for its inherent energy inefficiency.