Song selectivity and sensorimotor signals in vocal learning and production

Bird song, like human speech, is a learned vocal behavior that requires auditory feedback. Both as juveniles, while they learn to sing, and as adults, songbirds use auditory feedback to compare their own vocalizations with an internal model of a target song. Here we describe experiments that explore a role for the songbird anterior forebrain pathway (AFP), a basal ganglia-forebrain circuit, in evaluating song feedback and modifying vocal output. First, neural recordings in anesthetized, juvenile birds show that single AFP neurons are specialized to process the song stimuli that are compared during sensorimotor learning. AFP neurons are tuned to both the bird's own song and the tutor song, even when these stimuli are manipulated to be very different from each other. Second, behavioral experiments in adult birds demonstrate that lesions to the AFP block the deterioration of song that normally follows deafening. This observation suggests that deafening results in an instructive signal, indicating a mismatch between feedback and the internal song model, and that the AFP is involved in generating or transmitting this instructive signal. Finally, neural recordings from behaving birds reveal robust singing-related activity in the AFP. This activity is likely to originate from premotor areas and could be modulated by auditory feedback of the bird's own voice. One possibility is that this activity represents an efference copy, predicting the sensory consequences of motor commands. Overall, these studies illustrate that sensory and motor processes are highly interrelated in this circuit devoted to vocal learning, as is true for brain areas involved in speech.

Targeted deletion of the mouse POU domain gene Brn-3a causes selective loss of neurons in the brainstem and trigeminal ganglion, uncoordinated limb movement, and impaired suckling.

The Brn-3 subfamily of POU domain genes are expressed in sensory neurons and in select brainstem nuclei. Earlier work has shown that targeted deletion of the Brn-3b and Brn-3c genes produce, respectively, defects in the retina and in the inner ear. We show herein that targeted deletion of the Brn-3a gene results in defective suckling and in uncoordinated limb and trunk movements, leading to early postnatal death. Brn-3a (-/-) mice show a loss of neurons in the trigeminal ganglia, the medial habenula, the red nucleus, and the caudal region of the inferior olivary nucleus but not in the retina and dorsal root ganglia. In the trigeminal and dorsal root ganglia, but not in the retina, there is a marked decrease in the frequency of neurons expressing Brn-3b and Brn-3c, suggesting that Brn-3a positively regulates Brn-3b and Brn-3c expression in somatosensory neurons. Thus, Brn-3a exerts its major developmental effects in somatosensory neurons and in brainstem nuclei involved in motor control. The pheno-types of Brn-3a, Brn-3b, and Brn-3c mutant mice indicate that individual Brn-3 genes have evolved to control development in the auditory, visual, or somatosensory systems and that despite differences between these systems in transduction mechanisms, sensory organ structures, and central information processing, there may be fundamental homologies in the genetic regulatory events that control their development.

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine.

Cells of the exocrine pancreas produce digestive enzymes potentially harmful to the intestinal mucosa. Dopamine has been reported to protect against mucosal injury. In looking for the source of dopamine in the small intestine, we found that the duodenal juice contains high levels of dopamine and that the pancreas itself has a high dopamine [and dihydroxyphenylalanine (dopa)] content that does not change significantly after chemical sympathectomy. Furthermore, we were able to demonstrate tyrosine hydroxylase (TH) activity in control pancreas as well as in pancreas from rats after chemical sympathectomy. Immunostaining and in situ hybridization histochemistry confirmed both the presence of TH, dopamine, and the dopamine transporter, and the mRNAs encoding TH and dopamine transporter, and the presence of both types of vesicular monoamine transporters in the exocrine cells of the pancreas. Since there are no catecholaminergic enteric ganglia in the pancreas, the above results indicate that pancreatic cells have all the characteristics of dopamine-producing cells. We suggest that the pancreas is an important source of nonneuronal dopamine in the body, and that this dopamine has a role in protecting the intestinal mucosa and suggests that dopamine D1b receptor agonists might be used to help mucosal healing in the gastrointestinal tract.

Influence of general anesthetics on a specific neural pathway in Drosophila melanogaster.

The neural pathway that governs an escape response of Drosophila to sudden changes in light intensity can be artificially induced by electrical stimulation of the brain and monitored by electrical recording from the effector muscles. We have refined previous work in this system to permit reliable ascertainment of two kinds of response: (i) a short-latency response that follows from direct excitation of a giant fiber neuron in the interior of the fly brain and (ii) a long-latency response in which electrical stimulation triggers neurons in the optic ganglia that ultimately impinge on the giant fiber neuron. The general anesthetic halothane is reported here to have very different potencies in inhibiting these two responses. The long-latency response is obliterated at concentrations similar to those that cause gross behavioral effects in adult flies, whereas the short-latency response is only partially inhibited at doses that are 10-fold higher. Three other volatile anesthetic agents show a similar pattern. Thus, as in higher organisms, the Drosophila nervous system is differentiated into components of high and low sensitivity to general anesthetics. Moreover, this work shows that one of the sensitive components of the nervous system lies in the optic lobe and is readily assayed by its effect on downstream systems; it should provide a focus for exploring the effects of genetic alteration of anesthetic sensitivity.

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice.

Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BCM) that survive up to 3 weeks of age and exhibit several novel pathologic phenotypes including severe anemia, degeneration of neurons in the basal ganglia and brainstem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. In addition, SOD2m1BCM/SOD2m1BCM mice older than 7 days exhibit extensive mitochondrial injury within degenerating neurons and cardiac myocytes. Approximately 10% of SOD2m1BCM/SOD2m1BCM mice exhibit markedly enlarged and dilated hearts. These observations indicate that SOD2 deficiency causes increased susceptibility to oxidative mitochondrial injury in central nervous system neurons, cardiac myocytes, and other metabolically active tissues after postnatal exposure to ambient oxygen concentrations. Our SOD2-deficient mice differ from a recently described model in which homozygotes die within the first 5 days of life with severe cardiomyopathy and do not exhibit motor disturbances, central nervous system injury, or ultrastructural evidence of mitochondrial injury.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

Nerve growth factor in the anterior pituitary: localization in mammotroph cells and cosecretion with prolactin by a dopamine-regulated mechanism.

Nerve growth factor (NGF) is well characterized for its neurotrophic actions on peripheral sensory and sympathetic neurons and on central cholinergic neurons of the basal forebrain. Recent evidence, however, has shown high levels of NGF to be present in a variety of biological fluids after inflammatory and autoimmune responses, suggesting that NGF is a mediator of immune interactions. Increased NGF serum levels have been reported in both humans and experimental animal models of psychological and physical stress, thus implicating NGF in neuroendocrine interactions as well. The possible source(s) and the regulatory mechanisms involved in the control of serum NGF levels, however, still remain to be elucidated. We now report the presence of both NGF gene transcripts and protein in the anterior pituitary. Immunofluorescence analysis indicated that hypophysial NGF is selectively localized in mammotroph cells and stored in secretory granules. NGF is cosecreted with prolactin from mammotroph cells by a neurotransmitter-dependent mechanism that can be pharmacologically regulated. Activation of the dopamine D2 receptor subtype, which physiologically controls prolactin release, resulted in a complete inhibition of vasoactive intestinal peptide-stimulated NGF secretion in vitro, whereas the specific D2 antagonist (-)-sulpiride stimulated NGF secretion in vivo, suggesting that the anterior pituitary is a possible source of circulating NGF. Given the increased NGF serum levels in stressful conditions and the newly recognized immunoregulatory function of this protein, NGF, together with prolactin, may thus be envisaged as an immunological alerting signal under neuronal control.

Nonlinear control of heart rate variability in human infants.

Nonlinear analyses of infant heart rhythms reveal a marked rise in the complexity of the electrocardiogram with maturation. We find that normal mature infants (gestation greater than or equal to 35 weeks) have complex and distinctly nonlinear heart rhythms (consistent with recent reports for healthy adults) but that such nonlinearity is lacking in preterm infants (gestation > or = to 27 weeks) where parasympathetic-sympathetic interaction and function are presumed to be less well developed. Our study further shows that infants with clinical brain death and those treated with atropine exhibit a similar lack of nonlinear feedback control. These three lines of evidence support the hypothesis championed by Goldberger et al. [Goldberger, A.L., Rigney, D.R. & West, B.J. (1990) Sci. Am. 262, 43-49] that autonomic nervous system control underlies the nonlinearity and possible chaos of normal heart rhythms. This report demonstrates the acquisition of nonlinear heart rate dynamics and possible chaos in developing human infants and its loss in brain death and with the administration of atropine. It parallels earlier work documenting changes in the variability of heart rhythms in each of these cases and suggests that nonlinearity may provide additional power in characterizing physiological states.

Expression of protein encoded by varicella-zoster virus open reading frame 63 in latently infected human ganglionic neurons.

The ganglionic cell type in which varicella-zoster virus (VZV) is latent in humans was analyzed by using antibodies raised against in vitro-expressed VZV open reading frame 63 protein. VZV open reading frame 63 protein was detected exclusively in the cytoplasm of neurons of latently infected human trigeminal and thoracic ganglia. This is, to our knowledge, the first identification of a herpesvirus protein expressed during latency in the human nervous system.

Olfactory neuroblastoma is a peripheral primitive neuroectodermal tumor related to Ewing sarcoma.

Olfactory neuroblastoma (ONB) is a malignant tumor of the nasal mucosa whose histogenesis is unclear. A relationship to neuroblastoma (NB), a pediatric tumor of the sympathetic nervous system, is based on morphologic similarities and the expression of similar neural antigens. However, the clinical presentation of ONB differs from that of NB, and MYCN amplification characteristic of NB is not observed. We have therefore examined the relationship of this malignancy to other classes of neural tumors. In previous studies, two ONB cell lines demonstrated cytogenetic features and patterns of protooncogene expression suggestive of a relationship to the Ewing sarcoma family of childhood peripheral primitive neuroectodermal tumors (pPNETs). The pPNETs show t(11;22)(q24;q12) or t(21;22)(q22;q12) chromosomal translocations fusing the EWS gene from 22q12 with either the FL11 gene on 11q24 or the ERG gene on 21q22. We therefore analyzed ONBs for the presence of pPNET-associated gene fusions. Both cell lines showed rearrangement of the EWS gene, and fluorescence in situ hybridization (FISH) of each case demonstrated fusion of EWS and FL11 genomic sequences. Moreover, both lines expressed EWS/FL11 fusion transcripts with in-frame junctions between exon 7 of EWS and exon 6 of FL11 as described for pPNETs. We identified similar gene fusions in four of six primary ONB cases. None of the cases expressed tyrosine hydroxylase, a catecholamine biosynthetic enzyme widely expressed in NB. Our studies indicate that ONB is not a NB but is a member of the pPNET family.

Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation.

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.

High-affinity neuropeptide Y receptor antagonists.

Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.