Comprehensive Imaging of Sensory-Evoked Activity of Entire Neurons Within the Awake Developing Brain Using Ultrafast AOD-Based Random-Access Two-Photon Microscopy.

Determining how neurons transform synaptic input and encode information in action potential (AP) firing output is required for understanding dendritic integration, neural transforms and encoding. Limitations in the speed of imaging 3D volumes of brain encompassing complex dendritic arbors in vivo using conventional galvanometer mirror-based laser-scanning microscopy has hampered fully capturing fluorescent sensors of activity throughout an individual neuron's entire complement of synaptic inputs and somatic APs. To address this problem, we have developed a two-photon microscope that achieves high-speed scanning by employing inertia-free acousto-optic deflectors (AODs) for laser beam positioning, enabling random-access sampling of hundreds to thousands of points-of-interest restricted to a predetermined neuronal structure, avoiding wasted scanning of surrounding extracellular tissue. This system is capable of comprehensive imaging of the activity of single neurons within the intact and awake vertebrate brain. Here, we demonstrate imaging of tectal neurons within the brains of albino Xenopus laevis tadpoles labeled using single-cell electroporation for expression of a red space-filling fluorophore to determine dendritic arbor morphology, and either the calcium sensor jGCaMP7s or the glutamate sensor iGluSnFR as indicators of neural activity. Using discrete, point-of-interest scanning we achieve sampling rates of 3 Hz for saturation sampling of entire arbors at 2 µm resolution, 6 Hz for sequentially sampling 3 volumes encompassing the dendritic arbor and soma, and 200-250 Hz for scanning individual planes through the dendritic arbor. This system allows investigations of sensory-evoked information input-output relationships of neurons within the intact and awake brain.

Luminance Changes Drive Directional Startle through a Thalamic Pathway.

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Neural plasticity is affected by stress and heritable variation in stress coping style.

Here we use a comparative model to investigate how behavioral and physiological traits correlate with neural plasticity. Selection for divergent post-stress cortisol levels in rainbow trout (Oncorhynchus mykiss) has yielded low- (LR) and high responsive (HR) lines. Recent reports show low behavioral flexibility in LR compared to HR fish and we hypothesize that this divergence is caused by differences in neural plasticity. Genes involved in neural plasticity and neurogenesis were investigated by quantitative PCR in brains of LR and HR fish at baseline conditions and in response to two different stress paradigms: short-term confinement (STC) and long-term social (LTS) stress. Expression of proliferating cell nuclear antigen (PCNA), neurogenic differentiation factor (NeuroD) and doublecortin (DCX) was generally higher in HR compared to LR fish. STC stress led to increased expression of PCNA and brain-derived neurotrophic factor (BDNF) in both lines, whereas LTS stress generally suppressed PCNA and NeuroD expression while leaving BDNF expression unaltered. These results indicate that the transcription of neuroplasticity-related genes is associated with variation in coping style, while also being affected by STC - and LTS stress in a biphasic manner. A higher degree of neural plasticity in HR fish may provide the substrate for enhanced behavioral flexibility.

Gradients of ephrin-A2 and ephrin-A5b mRNA during retinotopic regeneration of the optic projection in adult zebrafish.

Regeneration of optic axons in the continuously growing optic system of adult zebrafish was analyzed by anterograde tracing and correlated with the mRNA expression patterns of the recognition molecules ephrin-A2 and ephrin-A5b in retinal targets. The optic tectum and diencephalic targets are all reinnervated after a lesion. However, the rate of erroneous pathway choices was increased at the chiasm and the bifurcation between the ventral and dorsal brachium of the optic tract compared to unlesioned animals. Tracer application to different retinal positions revealed retinotopic reinnervation of the tectum within 4 weeks after the lesion. In situ hybridization analysis indicated the presence of rostral-low to caudal-high gradients of ephrin-A2 and ephrin-A5b mRNAs in unlesioned control tecta and after a unilateral optic nerve lesion. By contrast, the parvocellular superficial pretectal nucleus showed retinotopic organization of optic fibers but no detectable expression of ephrin-A2 and ephrin-A5b mRNAs. However, a row of cells delineating the terminal field of optic fibers in the dorsal part of the periventricular pretectal nucleus was intensely labeled for ephrin-A5b mRNA and may thus provide a stop signal for ingrowing axons. Ephrin-A2 and ephrin-A5b mRNAs were not detectable in the adult retina, despite their prominent expression during development. Thus, given a complementary receptor system in retinal ganglion cells, expression of ephrin-A2 and ephrin-A5b in primary targets of optic fibers in adult zebrafish may contribute to guidance of optic axons that are continuously added to the adult projection and of regenerating axons after optic nerve lesion.

Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse.

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.

Expression of three distinct somatostatin messenger ribonucleic acids (mRNAs) in goldfish brain: characterization of the complementary deoxyribonucleic acids, distribution and seasonal variation of the mRNAs, and action of a somatostatin-14 variant.

In this study, three somatostatin (SRIF) complementary DNAs (cDNAs) were characterized from goldfish brain. The cDNAs encode three distinct preprosomatostatins (PSS), designated as PSS-I, PSS-II, and PSS-III. The goldfish PSS-I, PSS-II, and PSS-III contain enzymatic cleavage recognition sites, potentially yielding SRIF-14 with sequence identical to mammalian SRIF-14, SRIF-28 with [Glu1, Tyr7, Gly10]SRIF-14 at its C-terminus, and [Pro2]SRIF-14, respectively. The brain distribution of the three SRIF messenger RNAs (mRNAs) were differential but overlapping in the telencephalon, hypothalamus and optic tectum-thalamus regions. Seasonal variations in the levels of the three mRNAs were observed, with differential patterns between the three mRNAs and differences between the sexes. However, only the seasonal alteration in the levels of the mRNA encoding PSS-I showed close association with the seasonal variation in brain contents of immunoreactive SRIF-14 and inversely correlated with the seasonal variation in serum GH levels described in the previous studies, suggesting that SRIF-14 is involved in the control of the seasonal variation in serum GH levels. The putative SRIF-14 variant, [Pro2]SRIF-14, inhibited basal GH secretion from in vitro perifused goldfish pituitary fragments, with similar potency to SRIF-14; [Pro2]SRIF-14 also inhibited stimulated GH release from the pituitary fragments, supporting that [Pro2] SRIF-14 is a biologically active form of SRIF in goldfish.

Cyclic nucleotide-gated cation channel expression in embryonic chick brain.

Cyclic nucleotide-gated cation channels mediate sensory transduction in vertebrate photoreceptors and olfactory epithelium. These channels are also present in some non-sensory cells, but little is known of their physiological roles outside sensory systems. Using in situ hybridization we found that cyclic nucleotide channel mRNA is expressed specifically in the embryonic chicken forebrain, thalamus, optic tectum, basal midbrain and hindbrain, as well as in the branchial arches, limb buds and skin. Cyclic nucleotide gated channels may thus contribute to development or to cellular differentiation in the brain and in other tissues.

CalbindinD28k- and parvalbumin-immunoreactive neurons form complementary sublaminae in the rat superior colliculus.

By using light microscopic immunocytochemistry and computer analysis, we have mapped the distributions of two calcium-binding proteins (CaBPs), calbindinD28k (CB) and parvalbumin (PV), in the rat superior colliculus (SC). The patterns of CaBP expression were complementary. A band of heavily labeled, medium-sized CB-immunoreactive cells (CB-cells) was centered in the optic layer (OL), whereas PV-immunoreactive cells (PV-cells) were found predominantly in the intermediate gray layer (IGL), where they were clustered within patches of PV-labeled fibers. The superficial gray layer (SGL) could be divided into two sublaminae. CB-cells were found mostly in the dorsal half of the SGL, whereas PV-cells were scattered throughout the ventral SGL and the dorsal OL. Most of the CaBP-immunoreactive cells in the SGL were small bipolar cells with vertically oriented dendrites; however, there were also some PV-cells with horizontally oriented dendrites. Quantitative analysis of the CaBP distributions reinforced our observations that these cells are distributed in complementary tiers that are not restricted to the traditional laminae. The size and shape of some of these tiers were determined from a three-dimensional reconstruction of serial sections. The complementarity of the CaBP-immunoreactive tiers was also confirmed by fluorescence microscopy of double-labeled sections, in which few if any double-labeled neurons were observed. Complementary tiers of CB-cells and PV-cells have been observed previously in the SC of the cat. The present results demonstrate them in another species and further suggest that there are functional sublaminae in the SC that can be distinguished by CaBP content.

Localization of the Rev-ErbA orphan receptors in the brain.

In the present study, we report the localization of the Rev-ErbA alpha and beta nuclear orphan receptors, two closely related members of the nuclear hormone receptor superfamily, in the brain. Both Rev-ErbA variant mRNAs were highly expressed in the olfactory bulb, the hippocampus, and in the granular cells of the cerebellum, areas enriched also in other nuclear orphan receptors. Furthermore, the alpha-isoform was found in high amounts in the frontal cortex, the superficial gray layer of the superior colliculus, and the stria terminalis. Lower expression was observed in the nucleus accumbens, the caudate-putamen, and in some thalamic and brainstem nuclei. The beta-variant, in contrast, was only moderately expressed in the cortex, mainly in the striate and retrosplenial cortices. In addition, moderate levels of Rev-ErbA beta mRNA were seen in various thalamic, pontine and brainstem nuclei. We conclude that the two Rev-ErbA isoforms share a partly similar pattern of expression in the brain, especially in areas that also contain other nuclear orphan receptors and that otherwise the localization of the two receptor subtypes is differential.

Histamine-immunoreactive neurons and their innervation of visual regions in the cortex, tectum, and thalamus in the primate Macaca mulatta.

The histaminergic system is involved in the control of arousal in the brain and may impact significantly on visual processing. However, little is known about the histaminergic innervation of visual areas, or the histamine system in the primate brain, in general. We examined in Macaca mulatta the location of histamine-immunoreactive neurons and the innervation of important cortical and subcortical visual areas by histamine-immunoreactive axons. Brain sections were treated with an antibody to histamine and processed with standard immunohistological procedures. Histamine-immunoreactive neurons (20-45 microns in diameter) were localized bilaterally in the hypothalamus, particularly in ventral, lateral, posterior, and perimammillary hypothalamic areas. These hypothalamic cells appear to provide the sole neural source of histamine in the macaque brain. A plexus of varicose histamine-immunoreactive axons was present throughout the superior colliculus, the dorsal and ventral lateral geniculate nuclei of the thalamus, the reticular nucleus of the thalamus, the lateral posterior/pulvinar complex, and the visual cortex, including areas 17, 18, and the nearby extrastriate cortex. The axons nearly homogeneously innervated every region and layer in these structures, except for an increase in density in layer 1 of the visual cortex and in the superficial-most layers of the superior colliculus. Histaminergic axons broadly innervated every visual region examined. In comparison with the other aminergic and the cholinergic projection systems, which show considerable projection specificity, the histaminergic projection exhibited great homogeneity. The breadth of the distribution of histaminergic axons ensures that virtually all levels of visual processing in the primate can be influenced, either directly or indirectly, by the neuromodulatory effects of histamine.

GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits.

GABAA-receptors display an extensive structural heterogeneity based on the differential assembly of a family of at least 15 subunits (alpha 1-6, beta 1-3, gamma 1-3, delta, rho 1-2) into distinct heteromeric receptor complexes. The subunit composition of receptor subtypes is expected to determine their physiological properties and pharmacological profiles, thereby contributing to flexibility in signal transduction and allosteric modulation. In heterologous expression systems, functional receptors require a combination of alpha-, beta-, and gamma-subunit variants, the gamma 2-subunit being essential to convey a classical benzodiazepine site to the receptor. The subunit composition and stoichiometry of native GABAA-receptor subtypes remain unknown. The aim of this study was to identify immunohistochemically the main subunit combinations expressed in the adult rat brain and to allocate them to identified neurons. The regional and cellular distribution of seven major subunits (alpha 1, alpha 2, alpha 3, alpha 5, beta 2,3, gamma 2, delta) was visualized by immunoperoxidase staining with subunit-specific antibodies (the beta 2- and beta 3-subunits were covisualized with the monoclonal antibody bd-17). Putative receptor subtypes were identified on the basis of colocalization of subunits within individual neurons, as analyzed by confocal laser microscopy in double- and triple-immunofluorescence staining experiments. The results reveal an extraordinary heterogeneity in the distribution of GABAA-receptor subunits, as evidenced by abrupt changes in immunoreactivity along well-defined cytoarchitectonic boundaries and by pronounced differences in the cellular distribution of subunits among various types of neurons. Thus, functionally and morphologically diverse neurons were characterized by a distinct GABAA-receptor subunit repertoire. The multiple staining experiments identified 12 subunit combinations in defined neurons. The most prevalent combination was the triplet alpha 1/beta 2,3/gamma 2, detected in numerous cell types throughout the brain. An additional subunit (alpha 2, alpha 3, or delta) sometimes was associated with this triplet, pointing to the existence of receptors containing four subunits. The triplets alpha 2/beta 2,3/gamma 2, alpha 3/beta 2,3/gamma 2, and alpha 5/beta 2,3/gamma 2 were also identified in discrete cell populations. The prevalence of these seven combinations suggest that they represent major GABAA-receptor subtypes. Five combinations also apparently lacked the beta 2,3-subunits, including one devoid of gamma 2-subunit (alpha 1/alpha 2/gamma 2, alpha 2/gamma 2, alpha 3/gamma 2, alpha 2/alpha 3/gamma 2, alpha 2/alpha 5/delta).(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology of physiologically identified retinal X and Y axons in the cat's thalamus and midbrain as revealed by intraaxonal injection of biocytin.

Prior morphological studies of individual retinal X and Y axon arbors based on intraaxonal labeling with horseradish peroxidase have been limited by restricted diffusion or transport of the label. We used biocytin instead as the intraaxonal label, and this completely delineated each of our six X and 14 Y axons, including both thalamic and midbrain arbors. Arbors in the lateral geniculate nucleus appeared generally as has been well documented previously. Interestingly, all of the labeled axons projected a branch beyond thalamus to the midbrain. Each X axon formed a terminal arbor in the pretectum, but none continued to the superior colliculus. In contrast, 11 of 14 Y axons innervated both the pretectum and the superior colliculus, one innervated only the pretectum, and two innervated only the superior colliculus. Two of the Y axons were quite unusual in that their receptive fields were located well into the hemifield ipsilateral with respect to the hemisphere into which they were injected. These axons exhibited remarkable arbors in the lateral geniculate nucleus, diffusely innervating the C-laminae and medial interlaminar nucleus, but, unlike all other X and Y arbors, they did not innervate the A-laminae at all. In addition to these qualitative observations, we analyzed a number of quantitative features of these axons in terms of numbers and distributions of terminal boutons. We found that Y arbors contained more boutons than did X arbors in both thalamus and midbrain. Also, for axons with receptive fields in the contralateral hemifield (all X and all but two Y axons), 90-95% of their boutons terminated in the lateral geniculate nucleus; the other two Y axons had more of their arbors located in midbrain.

Neuropeptide Y innervation of retinorecipient layers of chick optic tectum.

A dense laminar network of varicose neuropeptide Y immunopositive fibres, but not cells, was found to cover the retinorecipient layers of the entire optic tectum of 10- to 21-day-old domestic chicks. Unilateral enucleation resulted in no apparent loss of neuropeptide Y immunopositive fibres in the contralateral tectum, suggesting that they are not of retinal origin. To identify possible sources of neuropeptide Y immunopositive tectal input, the distribution of neuropeptide Y immunopositive perikarya was investigated in the meso-diencephalic region of the chick. A virtually continuous network of neuropeptide Y immunopositive cells and fibres was observed stretching from rostro-lateral thalamus to the pretectum in close apposition to the perirotundal belt. These neuropeptide Y immunopositive structures did not seem to respect the borders of known anatomical regions but partially co-localized with the nucl. dorsolateralis anterior pars magnocellularis and pars medialis, nucl. pretectalis diffusus, nucl. lentiformis mesencephali pars parvocellularis and pars magnocellularis, nucl. principalis precommissuralis, nucl. lateralis precommissuralis, nucl. superficialis magnocellularis (SM), nucl. posteroventralis thalami Kühlenbeck and the nucl. subrotundus. In the nucleus of the basal optic root, neuropeptide Y immunopositive perikarya were observed only within or adjacent to its dorsal and lateral subdivisions although all subdivisions were enmeshed with neuropeptide Y immunopositive fibres. The neuropeptide Y immunopositive tectal input is likely to derive from tectothalamic--presumably perirotundal--neuronal groups. The extent of this tectal afferent projection, not reported earlier in the domestic chick, suggests a powerful neuropeptide-Yergic control of retinotectal relay function.

The distribution of excitatory amino acid binding sites in the brain of an electric fish, Apteronotus leptorhynchus.

The distribution of three types of exitatory amino acid receptors was examined in the brain of a high frequency weakly electric fish, Apteronotus leptorhynchus, by localizing the binding sites of ligands selective for mammalian kainic acid (KA), quisqualate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. All three binding sites were densest within the forebrain and in certain hypothalamic nuclei (nucleus tuberis anterior, inferior lobe). The core of the dorsal forebrain (dorsal centralis) had a very high density of NMDA binding sites and only moderate levels of AMPA and KA binding sites, while this was reversed for the dorsolateral forebrain. The AMPA and NMDA binding sites were found throughout the brain while KA binding sites were relatively restricted and were absent from most of the brainstem. The cerebellar molecular layer contained a very high density of KA and AMPA binding sites but almost no NMDA binding sites; the granular layer had a low density of AMPA and NMDA binding sites but was lacking in KA binding sites. All three types of binding sites were found within the electromotor system (nucleus electrosensorius and prepacemaker nucleus) at sites where the iontophoresis of glutamate causes species-specific behaviours. KA binding sites were found at only two sites along the electrosensory afferent pathways: (1) in the molecular layer of the electrosensory lateral line lobe, associated with a feedback pathway emanating from granule cells of the overlying cerebellum, and (2) in the lateral nucleus praeminentialis dorsalis, associated with a descending pathway emanating from the torus semicircularis. NMDA and AMPA binding sites are found throughout the electrosensory pathways. Within the electrosensory lateral line lobe the NMDA binding sites were predominantly associated with the feedback pathways terminating in its molecular layer and not with the deep neuropil layer containing primary electroreceptor afferents.

Differences in salmon GnRH and chicken GnRH-II contents in discrete brain areas of male and female rainbow trout according to age and stage of maturity.

We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2-10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.