Novel insights into the spatial and temporal complexity of hypothalamic organization through precision methods allowing nanoscale resolution.

The mammalian hypothalamus contains an astounding heterogeneity of neurons to achieve its role in coordinating central responses to virtually any environmental stressor over the life-span of an individual. Therefore, while core features of intrahypothalamic neuronal modalities and wiring patterns are stable during vertebrate evolution, integration of the hypothalamus into hierarchical brain-wide networks evolved to coordinate its output with emotionality, cognition and conscious decision-making. The advent of single-cell technologies represents a recent milestone in the study of hypothalamic organization by allowing the dissection of cellular heterogeneity and establishing causality between opto- and chemogenetic activity modulation of molecularly-resolved neuronal contingents and specific behaviours. Thus, organizational rules to accumulate an unprecedented variety of hierarchical neuroendocrine command networks into a minimal brain volume are being unravelled. Here, we review recent understanding at nanoscale resolution on how neuronal heterogeneity in the mammalian hypothalamus underpins the diversification of hormonal and synaptic output and keeps those sufficiently labile for continuous adaptation to meet environmental demands. Particular emphasis is directed towards the dissection of neuronal circuitry for aggression and food intake. Mechanistic data encompass cell identities, synaptic connectivity within and outside the hypothalamus to link vegetative and conscious levels of innate behaviours, and context- and circadian rhythm-dependent rules of synaptic neurophysiology to distinguish hypothalamic foci that either tune the body's metabolic set-point or specify behaviours. Consequently, novel insights emerge to explain the evolutionary advantages of non-laminar organization for neuroendocrine circuits coincidently using fast neurotransmitters and neuropeptides. These are then accrued into novel therapeutic principles that meet therapeutic criteria for human metabolic diseases.

Perineuronal and perisynaptic extracellular matrix in the human spinal cord.

Extracellular matrix (ECM) forms an active interface around neurons of the central nervous system (CNS). Whilst the components, chemical heterogeneity and cellular recruitment of this intercellular assembly in various parts of the brain have been discussed in detail, the spinal cord received limited attention in this context. This is in sharp contrast to its clinical relevance since the overall role of ECM especially that of its chondroitin sulphate-based proteoglycan components (CSPGs) was repeatedly addressed in neuropathology, regeneration, CNS repair and therapy models. Based on two post-mortem human specimen, this study gives the first and detailed description of major ECM components of the human spinal cord. Immunohistochemical investigations were restricted to the systematic mapping of aggrecan, brevican, proteoglycan link-protein as well as tenascin-R and hyaluronan containing matrices in the whole cranio-caudal dimension of the human spinal cord. Other proteoglycans like versican, neurocan and NG2 were exemplarily investigated in restricted areas. We show the overall presence of tenascin-R and hyaluronan in both white and grey matters whereas aggrecan, proteoglycan link-protein and brevican were restricted to the grey matter. In the grey matter, the ECM formed aggrecan-based perineuronal nets in the ventral and lateral horns but established single perisynaptic assemblies, axonal coats (ACs), containing link-protein and brevican in all regions except of the Lissauer's zone. Intersegmental differences were reflected in the appearance of segment-specific nuclei but not in overall matrix distribution pattern or chemical heterogeneity. Perineuronal nets were typically associated with long-range projection neurons including cholinergic ventral horn motorneurons or dorsal spinocerebellar tract neurons of the Clarke-Stilling nuclei. Multiple immunolabelling revealed that nociceptive afferents were devoid of individual matrix assemblies unlike glycinergic or GABAergic synapses. The detailed description of ECM distribution in the human spinal cord shall support clinical approaches in injury and regenerative therapy.

Perisynaptic aggrecan-based extracellular matrix coats in the human lateral geniculate body devoid of perineuronal nets.

The extracellular matrix surrounds different neuronal compartments in the mature nervous system. In a variety of vertebrates, most brain regions are loaded with a distinct type of extracellular matrix around the somatodendritic part of neurons, termed perineuronal nets. The present study reports that chondrotin sulfate proteoglycan-based matrix is structured differently in the human lateral geniculate body. Using various chondrotin sulfate proteoglycan-based extracellular matrix antibodies, we show that perisomatic matrix labeling is rather weak or absent, whereas dendrites are contacted by axonal coats appearing as small, oval structures. Confocal laser scanning microscopy and electron microscopy demonstrated that these typical structures are associated with synaptic loci on dendrites. Using multiple labelings, we show that different chondrotin sulfate proteoglycan components of the extracellular matrix do not associate exclusively with neuronal structures but possibly associate with glial structures as well. Finally, we confirm and extend previous findings in primates that intensity differences of various extracellular matrix markers between magno- and parvocellular layers reflect functional segregation between these layers in the human lateral geniculate body.

Chondroitin sulphate proteoglycan-based perineuronal net establishment is largely activity-independent in chick visual system.

Extracellular matrix components consisting of large aggregating chondroitin sulphate proteoglycans accumulate around neuronal perikarya to establish perineuronal nets. These perineuronal nets surround subpopulations of neurons in many vertebrates including man. In chickens, perineuronal nets show very fast matrix maturation after hatching which is probably due to the rapid establishment of neuronal morphology and immediate functional and behavioural performance of the animals. In mammals, maturation of extracellular matrix including perineuronal nets largely depends upon specific afferent activation. The present study shows that extracellular matrix maturation in mesencephalic, diencephalic and telencephalic visual centers of chicks tectofugal system is not principally determined by light activation. Perineuronal nets show an equally developed phenotypic character on monocularly light deprived animals in all investigated brain regions. Results suggest that establishment of extracellular matrix and perineuronal nets are largely activity-independent in the investigated precocial bird.

Enhanced Ras activity preserves dendritic size and extension as well as synaptic contacts of neurons after functional deprivation in synRas mice.

The monomeric GTP-binding protein p21Ras has been repeatedly implicated in neuronal stability and plastic changes of the adult nervous system. Recently, we have shown that expression of constitutively active Ras protein in transgenic synRas mice results in a significant increase in the dendritic size and complexity of differentiated pyramidal neurons as well as in increased synaptic connectivity. In the present study, we examined the organization of the vibrissae-barrel cortex in synRas mice and the effects of enhanced Ras activity on deprivation-induced dendritic reorganization after vibrissectomy. The results demonstrate a significant increase in vibrissae-barrel sizes and proportional spacing between barrels in synRas mice, suggesting that the neuronal target specificity of thalamocortical terminals is preserved. Accordingly, the arrangement of double bouquet cells at the borders of barrel columns ensuring functional distinctness is unchanged. Partial vibrissectomy is followed by significant dendritic regression of corresponding pyramidal neurons in the barrel cortex of wild-type mice, which, however, could not be observed in synRas mice. The results provide the first evidence for a role of Ras in preserving neuronal structure after functional deprivation in vivo.

Topographical organisation of projections from the nucleus isthmi magnocellularis to the optic tectum of the chick brain.

The anatomical connection of the magnocellular isthmic nucleus with the optic tectum was investigated with the axonal tracer biotinylated dextran amine. Following iontophoretic injection of this tracer into different areas of the chick optic tectum, neurones of both magno- and parvocellular isthmic nuclei were labelled together in a topographical arrangement. The number of labelled neurones in the parvocellular nucleus was generally higher than in magnocellular. Using different locations of the tracer injections, systematic shifts in the location of the labelled neurones were detected. The labelled axons were seen to course along the shortest possible distance between the injection site and the cells of origin, i.e., the ventral part of the tectum received projections from neurones located ventrally in the isthmic nuclei, the dorsal tectum from neurones in the dorsal part, and the lateral extension of the tectum from neurones lying midway along the nuclei. This parallel and topographic projection of the two nuclei was primarily observed in sagittal sections. After tracer injections into the magnocellular nucleus, the terminal arbours were seen to extend from the deep layers (11-12) to layer 2 of the tectum. The projections observed appeared to be topographically organised, and furthermore appeared to be parallel with and complimentary to previously described projections of the parvocellular isthmic nucleus.

The axon arbourisation of nuclei isthmi neurons in the optic tectum of the chick and pigeon. A Golgi and anterograde tracer-study.

The optic tectum is reciprocally connected to the nuclei isthmi pars magnocellularis (Imc) and pars parvocellularis (Ipc), which have different modulatory effects on optic transmission. We studied the axon arbourisation of these isthmic nuclei in the optic tectum in order to differentiate between them using Golgi-impregnated preparations both in chickens and pigeons. In addition, sections from animals injected with the anterograde tracer biotinylated dextran-amine (BDA) into the Imc were examined in the bright-field and electron microscope to identify the axon arbourisations and terminals. Also, GABA immunogold stained sections were examined in the electron microscope. In Golgi preparations, slab-like (or poplar tree-like) axon terminal arbourisations of both magnocellular and parvocellular isthmic nuclei neurons were found extending to the tectal surface, with similar branching patterns, but different lengths. The axon arbourisations extending from layer 5 of the optic tectum to the surface were termed type 1, whereas those extending from the internal (12-11) layers to the tectal surface were termed type 2. Type 2 arbourisations very closely matched arbourisations observed in BDA injected material, indicating that Imc neurons gave rise to type 2 arbourisations. The two kinds of axon arbourisation in the external tectal layers were alike in both types of bird, except for the width, which was about 10 mum larger in the type 2 axon arbour. Controlling for size, there was no significant difference between chicks and pigeons. The significance of these afferents in the optic tectum is discussed.

A comparative Golgi study of chicken (Gallus domesticus) and homing pigeon (Columba livia) hippocampus.

Neurons and fibres in the chick and homing pigeon hippocampus were described following Golgi impregnation. Two principal classes of neurons were distinguished: projection neurons with distant projecting axons and spiny dendrites, and local circuit neurons. In the homing pigeon and chicken hippocampus there are three types of projection neurons: pyramidal, pyramidal-like and multipolar. The pyramidal and pyramidal-like neurons are only found in the central 'pyramidal' layer of the hippocampus whereas multipolar neurons are present in the suprapyramidal, pyramidal and infrapyramidal layers. The axon of projection neurons typically emits several varicose collaterals from the initial section. Most of these collaterals extend along the infrapyramidal layer of the hippocampus, while others ascend to the pyramidal and suprapyramidal layers where they branch. The number of impregnated axon collaterals was higher in the homing pigeon than in the chick hippocampus. A variety of multi-angular/ovoid local circuit neurons ranging from small to large size are found in the homing pigeon and chick hippocampus. Their axons develop local arborization of varicose branches, the extent of which varies with the type of local circuit neurons. The density of GABA immunopositive local circuit neurons was found to be greater in the homing pigeon than in the chick. The profuse arborization of projection neuron axon collaterals and the higher density of GABA-immunopositive local circuit neurons in the homing pigeon hippocampus may underlie the differences in hippocampal function between the homing pigeon and chick, and this complex local connectivity may contribute to the ability of spatial orientation and memory.

A golgi and a combined Golgi/GABA immunogold study of local circuit neurons in the homing pigeon hippocampus.

Three types of local circuit neurons have recently been reported in the homing pigeon hippocampus. The principal type appears to be constituted by the medium-sized angular or ovoid local circuit neurons that occur in all layers of the hippocampus. The current Golgi study has revealed that these neurons can be classified according to their axonal arborisation extension: (1) in all directions, (2) principally medio-laterally, or (3) dorso-ventrally. The local circuit neurons with dorso-ventral axon arborisation are present only in the subpyramidal layer. Serial sections of a Golgi-impregnated medium-sized, multiangular local circuit neuron in the pyramidal layer and a small, ovoid neuron in the suprapyramidal layer were investigated in the electron microscope. Some of these sections were processed for GABA immunogold cytochemistry. The soma and large dendrites of both neurons displayed GABA immunogold labelling. On the soma of medium-sized local circuit neuron there were numerous terminals; on the soma of the small one relatively fewer terminals were observed. The terminals contained round and/or flat synaptic vesicles. The long axonal branches of the neurons exhibited varicosities containing flattened or pleomorphic vesicles. Axo-dendritic, axo-somatic and a few axo-axonic synapses were observed. The large dense axon arborisation field of medium-sized local circuit neurons is properly situated to modulate intrinsic hippocampal activity and that of the small local circuit neurons is well situated to modulate the hippocampal input in the suprapyramidal layer.

Efferent connections of the ectostriatal core. An anterograde tracer study.

In the present study the efferent connections of the ectostriatal core were investigated with biotinylated dextran-amine anterograde tracer in the chicken using light microscopy. The efferents of the ectostriatal core were labelled anterogradely, but retrograde labelling was also observed, which displayed the afferents of the same region. The ectostriatal belt received a few thin varicose fibres; retrogradely labelled cells also appeared. The most extended projection ended in the surrounding neostriatum, which turned out to be reciprocally connected to the ectostriatal core. On the basis of these connections, the neostriatum is said to be an important visual associative center. Efferent fibres reached the motor areas as well. A significant projection entered the paleostriatum augmentatum, especially the ventral part. The paleostriatum primitivum also received a few fibres. The other motor center, the medial part of the anterior archistriatum, was proved to be directly connected to the ectostriatal core as well. Considering that the archistriatum is also connected indirectly to the Wulst, the movements are able to be guided by well processed visual information.

Electron microscopic data on the neurons of nuclei subpretectalis and posterior-ventralis thalami. A combined immunohistochemical study.

The nucleus rotundus receives GABA-like immunoreactive fibres from the nuclei subpretectalis and postero-ventralis thalami. This result was confirmed by Phaseolus vulgaris leucoagglutinin (PhA-L) anterograde tracer and with electron microscopic (EM) gamma-aminobutiric acid (GABA)-immunogold staining. The detailed electron microscopic analysis of the structure of the neurons in these nuclei revealed that the neurons in the nucleus subpretectalis displayed GABA-like immunoreactivity. In the postero-ventral thalamic nucleus a group of neurons was GABA-positive. The surface of the neurons was covered both with numerous GABA-negative and GABA-like immunoreactive terminals that established asymmetrical and symmetrical synapses, respectively, with the GABA-positive neurons. The GABA-like immunonegative terminals are supposed to be the axon terminals of the collaterals of tecto-rotundal fibres in the subpretectal nucleus and the collateral terminal branches of contralateral tecto-rotundal fibres in the postero-ventralis thalami. In both nuclei, the GABA-like immunoreactive terminals may be developed by the collaterals of local neurons that establish symmetrical synapses. In the Phaseolus lectin-stained preparations these terminals may be labelled. The morphological characteristics of the neurons in the subpretectal and partly, in the posteroventral nuclei are similar to those of interneurons (local circuit neurons) and the numerous asymmetrical and symmetrical axo-somatic synapses, respectively. But these neurons locate outside of their target nucleus, and exert their modulatory effect on rotundo-ectostriatal transmission. Also, a contralateral influence is present in the nucleus rotundus that may interact in the cooperation of the eyes. The neurons of the subpretectal and posteroventral nuclei, similarly to the neurons of isthmic nuclei, are a special group of modulatory neurons with effects at a distance.

Telencephalic connections of the visual system of the chicken: tracing the interrelation of the efferents of the visual Wulst and the hyperstriatum ventrale.

In the present study the telencephalic connections between the centres of the tectofugal and thalamofugal pathways were investigated with Phaseolus vulgaris leucoagglutinin and biotinylated dextran-amine anterograde tracers in chicken using light and electron microscopy. No direct connection was found between the visual Wulst and the ectostriatum or the telencephalic centres of the tectofugal and thalamofugal pathways. Besides other projections, the visual Wulst emitted fibres also to the middle and lateral parts of the hyperstriatum ventrale. Further experiments revealed that the middle part of the hyperstriatum ventrale projected to the ectostriatum centrale and periphericum and established an indirect connection between the visual Wulst and the ectostriatum. The lateral part of the hyperstriatum ventrale sent a few efferent fibres toward the diencephalon and brainstem, but projected massively to the ectostriatum periphericum, neostriatum intermedium pars laterale, the ventral part of the neostriatum caudale and the archistriatum dorsale. Considering that these areas are structures of the tectofugal circuitry as well, the presence of these connections may result in a more elaborate visual processing. The neostriatum may be an associative visual center and possibly a modulatory area toward the archistriatum intermedium dorsale, by which visual information may mediate, modulate and control the movements.

Comparison of Healon and Viscoat in cataract extraction and intraocular lens implantation.

Sixty patients were randomly assigned to Healon (20 patients) or Viscoat (40 patients) treatment during extracapsular cataract extraction and intraocular lens implantation surgery. The 40 patients in the Viscoat group were randomly subdivided into two groups. In one group (20 patients), Viscoat was irrigated/aspirated from the eye at the close of surgery, while in the second group of 20 patients, Viscoat was left in the eye. In all Healon cases, the viscoelastic substance was removed from the eye at the end of the surgical procedure. Compared with Viscoat, Healon better facilitated the surgical procedure and appeared to be a more advantageous viscoelastic preparation. Viscoat, in many cases, caused rises in intraocular pressure in the immediate postoperative period when either removed or left in the eye at the close of surgery.

Botulinum toxin and its uses in the treatment of ocular disorders.

The recent utilization of type A botulinum toxin in clinical ophthalmology makes it important for all health professionals involved in eye care to understand the action of this "non-surgical" tool. This paper summarizes the present knowledge concerning the chemistry, toxicology, and pathophysiology of the toxin. It also discusses the indications, contraindications, safety, and efficacy of this use of botulinum toxin.

Epidermal growth factor receptors on corneal endothelium.

If a growth factor could bind to and stimulate human endothelial healing, corneal disease could be minimized. To this end, primary cultures of feline and human corneal endothelium were tested in receptor binding assays for radiolabeled epidermal growth factor (EGF). Both of these cells bound ten times as much 125I-EGF as did the negative control cell lines. The time course of association of 125I-EGF to cat corneal endothelium was found to be complete after approximately 120 minutes at 22 degrees C. The 125I-EGF was shown not to dissociate greatly when fresh binding buffer was added to endothelial cultures that had bound the radiolabeled peptide. The pH optimum for binding was determined to be approximately 6.4. The receptor number per cell and the affinity constant for binding were determined to be 40,000 receptors per cell and 1.1 x 10(9) L/mole, respectively, using a Scatchard plot. Parallel cultures of human fetal corneal endothelium grew in vitro only when the growth medium was supplemented with low concentrations of EGF. These studies provide evidence that EGF is specifically bound to the corneal endothelium.