Herpes simplex virus as a transneuronal tracer.

Determining the connections of neural systems is critical for determining how they function. In this review, we focus on the use of HSV-1 and HSV-2 as transneuronal tracers. Using HSV to examine neural circuits is technically simple. HSV is injected into the area of interest, and after several days, the animals are perfused and processed for immunohistochemistry with antibodies to HSV proteins. Variables which influence HSV infection include species of host, age of host, titre of virus, strain of virus and phenotype of infected cell. The choice of strain of HSV is critically important. Several strains of HSV-1 and HSV-2 have been utilized for purposes of transneuronal tract-tracing. HSV has been used successfully to study neuronal circuitry in a variety of different neuroanatomical systems including the somatosensory, olfactory, visual, motor, autonomic and limbic systems.

Neurons that migrate from the olfactory epithelium in the chick express luteinizing hormone-releasing hormone.

In several mammalian species, luteinizing hormone-releasing hormone (LHRH) neurons have been shown to migrate from nasal regions to the brain during early development. Using immunocytochemistry, we have identified LHRH containing neurons in developing chick embryos. In embryonic day 4 (E4) and E5 animals, a small group of LHRH immunoreactive (IR) neurons were found just ventral to the olfactory pit. LHRH-IR neurons were also found within the immunoreactive (IR) neurons were found just ventral to the olfactory pit. LHRH-IR neurons were also found within the nasal epithelium. In E6 and E7 animals, many more LHRH-IR neurons were observed in nasal epithelium, in close association with the olfactory nerve, and within the telencephalon. These data are consistent with the hypothesis that LHRH neurons in chicks originate within nasal structures and migrate into the brain.

Cerebrospinal fluid of Parkinson's disease patients inhibits the growth and function of dopaminergic neurons in culture.

We report the possible existence of an inhibitory factor in the CSF of Parkinson's disease patients that inhibits the function and growth of dopaminergic neurons in rat mesencephalic culture. After 40 hours' exposure to the < 10 kd fraction of CSF from PD patients, the high-affinity dopamine uptake was 66% of that of cultures exposed to CSF from controls. However, the number of dopaminergic neurons remained unchanged at this time. After 90 hours' exposure to the < 10 kd fraction of CSF from PD patients, the number of dopaminergic neurons decreased to 10% of that in cultures exposed to CSF from controls, and the size of the remaining dopaminergic neurons in the culture became smaller. This inhibitory factor did not affect the growth of other types of neurons. The chemical nature of this inhibitory factor is under investigation.

Survival and functional demonstration of interregional pathways in fore/midbrain slice explant cultures.

An important general question in neurobiology concerns the development and expression of the rich context of neuronal phenotypes, especially in relation to the diverse patterns of connectivity. Organotypic cultures of brain slices may offer distinct advantages for such studies if such a preparation survives, maintains a wide diversity of neuronal phenotypes and displays appropriate synaptic connections between regions. To address these requirements, we utilized long-term organotypic cultures of intact horizontal slices of rat forebrain and midbrain and assessed a variety of markers of phenotype in combination with functional tests of connectivity. This explant preparation displayed a distinct viability requirement such that the greatest explant survival was seen in slices taken from pups of less than postnatal day 7 and was independent of N-methyl-D-aspartate channel blockade. The anatomical features of the major brain regions (e.g., neocortex, striatum, septum, hippocampus, diencephalon and midbrain) were observed in their normal boundaries. The presence of cholinergic and catecholaminergic neurons was demonstrated with acetylcholinesterase histochemistry and tyrosine hydroxylase immunohistochemistry. Labelled neurons displayed multiple, regionally-appropriate cytoarchitectures and, in some cases, could be seen to project to brain regions in a manner quite similar to that seen in vivo. Finally, the direct demonstration of spontaneous and evoked interregional excitatory synaptic transmission was made using whole-cell patch-clamp recordings from striatal neurons which revealed an intact glutamate-using corticostriatal pathway. This simple explant preparation appears to contain a rich diversity of neuronal types and synaptic organization. Therefore, this preparation appears to have several distinct advantages for basic neurobiologic research since it combines long-term culture viability and many features of mature brain including complex interregional neuronal systems.

A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethylbenzidine as markers.

Techniques for correlative double-label immunocytochemistry (ICC) at light and electron microscopic (EM) level are useful for determining the neurotransmitter phenotype of inputs onto immunocytochemically identified neurons. Tetramethylbenzidine (TMB) has been used as a chromogen at the EM level for horseradish peroxidase tract tracing. We have found that TMB, in combination with diaminobenzidine (DAB), can be used in a double-label immunocytochemical protocol to examine neuropeptide Y inputs onto luteinizing hormone-releasing hormone cells in the sheep preoptic area. At both light and EM levels, TMB reaction product is visibly distinct from DAB reaction product. The ultrastructural preservation we have been able to obtain with our technique is better than that obtained with techniques that use TMB at a lower pH. Furthermore, this technique allows the demonstration of synaptic contacts between neurochemically identified terminals and cells with different neurotransmitter phenotypes.

Retrograde transneuronal transport of herpes simplex virus in the retina after injection in the superior colliculus, hypothalamus and optic chiasm.

Immunocytochemistry was used to identify infected cells after injection of Herpes simplex virus (HSV) into the superior colliculus, hypothalamus and optic chiasm. Ganglion cells of the retina were labeled in a pattern consistent with known projections to retinorecipient nuclei. Cells of both the inner and outer nuclear layer were labeled. If this represents retrograde transneuronal transport, then HSV may provide an important tool for studying the neuronal circuitry of the retina.

The direction of apomorphine-induced rotation behavior is dependent on the location of excitotoxin lesions in the rat basal ganglia.

Adult rats received unilateral kainic acid (KA) lesions of the striatum with the anterior/posterior coordinates of the lesion at either 1.5 mm or 0.3 mm anterior to bregma. Four to six weeks after the lesion rats were placed in an open field environment and injected with apomorphine (1 mg/kg, s.c.). Rats receiving the more posterior lesion (0.3 mm) rotated ipsilateral to the lesioned side of the brain. In contrast, the majority of rats receiving the more anterior (1.5 mm) placement of the lesion rotated contralateral to the lesioned side of the brain. Histological analysis of several animals receiving posterior lesions revealed damage to the hippocampus and thalamus that was not seen in the animals receiving anterior lesions. Our results are consistent with the hypothesis that the direction of apomorphine-induced rotation after excitotoxin injection into the rat basal ganglia is dependent on the location of the lesion.

Magnetic resonance imaging of neural transplants in rat brain using a superparamagnetic contrast agent.

Rat fetal brain tissue was incubated in vitro with superparamagnetic ferrite particles covalently coupled to the lectin wheat germ agglutinin (WGA) and transplanted into the adult rat striatum. At 6 days and at 3 weeks post-surgery the transplants were observed on T1 weighted magnetic resonance (MR) images of the rat head as an area of relatively low signal intensity which could be clearly differentiated from the higher signal intensity produced by the host brain. Histological analysis revealed that the ferrite particles were largely restricted to the transplant in a patchy distribution. The ferrite particles were associated with cells having an apparent normal morphology. Superparamagnetic ferrite particles act as potent MR contrast agents and can be used to label transplanted cells. The labeled cells are apparently not adversely affected by the WGA-ferrite particles and can be monitored for at least three weeks in vivo using noninvasive MR imaging.

Sensitization of c-fos expression in rat striatum following multiple challenges with D-amphetamine.

D-Amphetamine transiently stimulates the expression of the immediate-early response gene, c-fos, in rat striatal cell nuclei. D-Amphetamine (2.5 mg/kg i.p.) induced a significantly greater expression of Fos-like immunoreactivity in striatum of rats treated three days previously with D-amphetamine compared to rats treated three days previously with saline. This increase in the expression of Fos-like immunoreactivity in rat striatum was characterized by a significantly greater number of immunoreactive nuclei and a significant increase in the intensity of the immunoreactivity. This sensitization of c-fos expression following a repeated administration of D-amphetamine indicates an increased activation of post-synaptic elements in rat striatum.

Subpopulations of migrating neurons express different levels of LHRH in quail and chick embryos.

LHRH neurons of the septal-preoptic area originate in the olfactory placode and migrate in the olfactory nerve into the brain during embryonic development. In adult birds, LHRH neurons have been found in the septal-preoptic area, mesencephalon and more recently in the lateral anterior nucleus of the thalamus (LA). LHRH neurons of the LA do not originate in the olfactory placode. Using immunocytochemistry, we examined the distribution of LHRH neurons in the embryonic and adult quail nervous system. The pattern of LHRH immunostaining in quail embryos was similar to that seen in chick embryos. However, there were many fewer neurons immunostained for LHRH from the olfactory placode to the septal-preoptic area in quail than in chick embryos. In contrast, there were more labeled neurons and more intense LHRH immunostaining in the thalamus of the quail than in the thalamus of chick embryos. In agreement with other studies, our data suggest that there are species differences in LHRH expression in migrating neurons. The current results should also be considered for quail-chick chimeras involving the olfactory placode.

Luteinizing hormone-releasing hormone in the pigeon terminal nerve and olfactory bulb.

The presence of a terminal nerve in the avian brain has recently been reported. As the terminal nerve in other classes of vertebrates contains luteinizing hormone-releasing hormone (LHRH), we used immunocytochemistry to determine whether the pigeon terminal nerve also contained LHRH. We found LHRH-immunoreactivity in the olfactory nerve and in the olfactory bulb. The distribution of LHRH neurons was similar to the LHRH neuronal migration pathway during development.

FUSE-binding protein is developmentally regulated and is highly expressed in mouse and chicken embryonic brain.

GAP-43 modulates axon guidance and neuronal plasticity. In vitro, FUSE-binding protein (FBP) binds to a segment of GAP-43 mRNA which regulates the stability of the transcript. FBP has also been shown to bind to a c-myc cis element and regulate transcription. In the current work, analysis of RNA and protein expression indicated that FBP is expressed in a distinct spatial temporal pattern during embryonic development. Expression was particularly high in the brain. In the adult, expression was not detected in most tissues but was still prominent in the brain and teste. This finding is consistent with a dual role of the protein as a single-strand polynucleotide-binding protein.

Tangential migration of luteinizing hormone-releasing hormone (LHRH) neurons in the medial telencephalon in association with transient axons extending from the olfactory nerve.

During embryonic development, luteinizing hormone-releasing hormone (LHRH) neurons migrate to the brain from the medial olfactory epithelium through the olfactory nerve. LHRH neurons enter the brain and migrate tangentially along the medial edge of the telencephalon in close association with a neural cell adhesion molecule (N-CAM) enriched fiber bundle. In the current work we wished to determine whether this N-CAM enriched fiber bundle is an extension of the olfactory nerve. Ablation experiments, immunocytochemistry and diI implants all suggest that LHRH neurons migrate in association with a very small subset of transient N-CAM enriched neuronal processes which extend out of the olfactory nerve proper to the septal-preoptic area.

A new chromogen for use in HRP-tract tracing and double-label immunocytochemistry.

A new chromogen, Indophane Blue (IB) was tested for use in HRP-tract tracing and double-label immunocytochemistry. Although TMB was slightly more sensitive and delineated individual labeled fibers better than IB in tract-tracing experiments, there were two advantages to using IB over TMB. First, the IB reaction could be performed at a pH of 6.0, rather than the pH of 3.3 required for TMB, resulting in much better preservation of tissue. Second, there was no crystalline artifact as has been observed with TMB. In the double-label immunocytochemistry experiments, the combination of diaminobenzidine for the first chromogen and IB as the second, provided excellent contrast.

Single- and double-label immunocytochemical study of the ovine suprachiasmatic nucleus (SCN): GABAergic and peptidergic relationships.

This study evaluated the neuropeptide and neurotransmitter content of the ovine suprachiasmatic nucleus (SCN) using both single- and double-label immunocytochemical methods. Single-label immunocytochemistry identified a few lightly labeled gamma aminobutyric acid (GABA) cells within the SCN as well as a dense plexus of fibers staining positive for the GABA biosynthetic enzyme, glutamic acid decarboxylase (GAD). Vasoactive intestinal polypeptide (VIP) fibers exhibited a similar distribution to GAD fibers; VIP cells were found throughout the SCN, as well as in the paraventricular (PVN) and supraoptic nuclei. Both GAD and VIP fibers exited dorsally from the SCN towards the PVN. Neurophysin (NP) and neuropeptide-Y (NPY) fibers were sparsely distributed throughout the SCN. Double-label immunocytochemistry revealed that GAD varicosities were often in close apposition to VIP cells. These results confirm the presence of GABAergic elements within the sheep SCN. Furthermore, they raise the possibility of a GABAergic modulation of VIP neuronal activity within the ovine SCN.

Anterograde transport of HSV-1 and HSV-2 in the visual system.

The anterograde spread of herpesvirus in the visual system subsequent to retinitis has been observed clinically. We compared the ability of two well-studied Herpes simplex virus (HSV) strains to be transported in the anterograde direction in the hamster visual system: strain McIntyre, representing HSV-1, and strain 186, representing HSV-2. Intravitreal injection of HSV-2 labeled more retinorecipient neurons than did HSV-1, suggesting important type differences in the ability of HSV to infect retinorecipient neurons after intravitreal injection. The most likely explanation for our results is that HSV-2 is more efficiently adsorbed than HSV-1 in the retinal ganglion cells. Our results also suggest that HSV may be useful as an anterograde transneuronal tracer for neuroanatomical studies of the visual system.

Nuclear transfer technology in mammalian cloning.

The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

Disruption of the olfactory placode and brain conditioned medium increase the number of LHRH immunostained neurons in explants.

The olfactory placode gives rise to both olfactory receptor neurons, which remain as a component of the peripheral nervous system, and to luteinizing hormone-releasing hormone (LHRH) neurons, which migrate to the central nervous system. In this study, we used chick olfactory placode explants to ask several questions regarding LHRH neuronal differentiation. We found that explants of ectoderm from the fronto-nasal region of embryos as early as Hamilton & Hamburger (HH) stage 12 gave rise to LHRH neurons, that explants from all regions of the olfactory placode were able to generate LHRH neurons, that both brain conditioned medium and disruption of the olfactory placode increase the number of LHRH neurons observed in explants, and that the combination of these two manipulations results in the production of more LHRH neurons than either treatment alone. We conclude that LHRH neurons originate in the olfactory epithelium and that some of the same factors which influence olfactory receptor neuron development also affect LHRH neuronal development.

Citric acid-ammonium acetate buffer.

A pH table is reported for citric acid-ammonium acetate buffers that are useful for horseradish peroxidase (HRP) histochemistry.