Cerebral white matter damage in HIV infection demonstrated using beta-amyloid precursor protein immunoreactivity.

We have examined brain sections from 55 autopsy cases of AIDS for the prevalence and severity of axonal damage, assessed using beta-amyloid precursor protein (beta APP) immunoreactivity as a marker of such damage. The cases were subdivided into cases with HIV encephalitis with multinucleated giant cells (MGC), cases with other specific pathology, such as cerebral toxoplasmosis or lymphoma, cases with non-specific pathology and cases with no pathology. Significantly more foci containing beta APP+ axons were found in cases with HIV encephalitis with MGC (80%) and in cases with other specific pathology (58%) than in those with non-specific (30%) or no pathology (30%). The prevalence and abundance of beta APP+ axons generally paralleled the severity of pallor of myelin staining of cerebral white matter in cases without other specific pathology but in 4 cases without any pallor of myelin staining beta APP+ axons were present, suggesting that it may be a more sensitive marker of some forms of white matter damage in HIV infection than myelin pallor. Foci of beta APP+ axons were found in subcortical and deep white matter but did not convincingly co-localise with foci of demonstrable HIV infection as indicated by the presence of MGC and HIV p24 immunoreactivity. In contrast, they showed an approximately perivascular distribution at some sites in all of the disease categories studied. We consider this localisation to be more suggestive of a vascular pathogenetic mechanism of deep white matter damage in HIV infection than a mechanism dependent on diffusion of local myelinotoxic products from foci of cerebral HIV infection.

A novel protein, amyloid precursor-like protein 2, is present in human brain, cerebrospinal fluid and conditioned media.

A monoclonal antibody, 3B11, was raised to a novel protein, amyloid precursor-like protein 2, which did not recognize amyloid precursor protein. Multiple bands were detected in human brain fractions and cell lysate by Western blotting, indicating the presence of isoforms, 3B11 immunoreactivity was also detected in cerebrospinal fluid and conditioned medium, indicating that the protein is secreted. Immunocytochemistry revealed 3B11 immunoreactivity in sections of human brain.

Ubiquitinated inclusions in inclusion-body myositis patients are immunoreactive for cathepsin D but not beta-amyloid.

The nature of the inclusions in the human muscle disease inclusion-body myositis (IBM) has been the subject of debate. Parallels with Alzheimer's disease have been drawn after these inclusions were found to be ubiquitinated, and immunoreactive with antibodies to beta-amyloid (A beta) and certain amyloid-associated proteins. We have used a battery of antibodies against A beta and associated proteins to immunostain muscle biopsies from patients with IBM. Although the inclusions are ubiquitinated, we could not show immunoreactivity for A beta or the associated proteins investigated. We did, however, find that the ubiquitinated inclusions colocalised with the lysosomal marker, cathepsin D.

Non-Alzheimer neurofibrillary tangles show beta-amyloid-like immunoreactivity.

As well as being a neuropathological hallmark of Alzheimer's disease (AD), neurofibrillary tangles (NFT) are present in the brain in a number of other neurodegenerative conditions. beta-Amyloid (beta/A4) plaque formation is a central event in AD, although recent reports indicate that beta/A4, and its precursor protein (beta APP), are also associated with AD NFT. Using an antibody to a beta APP epitope overlapping the beta/A4 region, we demonstrate that immunoreactivity is found in extracellular NFT in Niemann-Pick disease, Hallervorden-Spatz disease, progressive supranuclear palsy and subacute sclerosing panencephalitis. These NFT were not labelled with a beta/A4 antibody which recognizes plaque beta/A4 in AD, which supports the proposal that beta/A4 can exist in different conformations. Our results also imply a common mechanism of NFT development in different neurodegenerative conditions, including those not normally associated with substantial beta/A4 deposition or beta APP involvement.

Microwave antigen retrieval of beta-amyloid precursor protein immunoreactivity.

Formalin fixation reduces beta-amyloid precursor protein (beta APP) immunoreactivity which restricts its study in archival tissue. Formic acid pretreatment has previously been used in an attempt to overcome this problem, but makes the sections very friable. In the present study, a microwave antigen retrieval method has been compared with formic acid pretreatment for retrieving beta APP immunoreactivity in formalin-fixed, paraffin-embedded human brain tissue. Microwave treatment resulted in superior retrieval of beta APP antigenicity in dystrophic neurites in Alzheimer's disease and in injured axons after head injury, using antibodies to three different epitopes. Unlike formic acid, microwave treatment causes minimal adverse effects on the strength and slide adhesion of the sections.

The paragigantocellular nucleus of the ventral medulla: a secondary source of cholinergic innervation of rat brainstem nuclei.

Many parts of the brainstem are known to be innervated by the cholinergic neurons of the pontomesencephalic tegmentum, but other possible sources of this innervation have rarely been considered. We sought to examine whether other cells in the brainstem were responsible for this cholinergic input using axonal tract tracing and choline acetyltransferase (ChAT) immunocytochemistry. The results confirm previous studies on the projections of the neurons of the pontomesencephalic tegmentum but also show that a group of ChAT-positive cells in the paragigantocellular nucleus in the ventral medulla are a source of widespread, albeit less substantial cholinergic projections to several areas of the brainstem.

Cholinergic neurons in the ventral trapezoid nucleus project to the cochlear nuclei in the rat.

A combination of retrograde axonal tract tracing and choline acetyltransferase immunocytochemistry was used to determine the cells of origin of the cholinergic innervation of the rat cochlear nucleus. The results showed that the cochlear nucleus receives a major cholinergic input from a group of small cells found in the ventral trapezoid nucleus. Experiments using an anterograde tracer confirmed the presence of a neuronal pathway from the ventral trapezoid nucleus to the cochlear nucleus and showed that this pathway travels via the trapezoid body.

A cholinergic propriospinal innervation of the rat spinal cord.

Previous work has suggested the presence of a widespread, intrinsic cholinergic innervation of the spinal cord. A combination of retrograde axonal tract tracing and choline acetyltransferase immunocytochemistry was used to show that there is a propriospinal cholinergic innervation of the rat spinal cord that arises from short-range projections (spanning up to six spinal segments) of cholinergic neurons known as 'central canal' and 'partition' cells.

Markers of axonal injury in post mortem human brain.

beta-Amyloid precursor protein (beta APP) can be detected immunocytochemically at sites of axonal injury in the brain, and has recently been found to be a useful marker for injured axons in patients who survived for only 3 h after head trauma. It is transported by fast axonal transport and is thought to accumulate in detectable levels where the cytoskeleton breaks down. If this theory is correct, other substances should accumulate here in the same way, so we have used antibodies to other neuronal proteins to compare their efficacy as markers of axonal injury. SNAP-25, chromogranin A and cathepsin D also marked injured axons at all survival times studied (2.5 h-2 weeks), although they were not as sensitive or specific as beta APP. Immunolabelling for the 68-kDa neurofilament subunit (NF68) was present in most uninjured axons, and allowed axonal swellings to be seen in some cases. Synaptophysin, GAP-43, ubiquitin or tau did not label any normal or injured axons in this study. We, therefore, suggest that beta APP should be the immunocytochemical marker of choice for the detection of injured axons. This study also showed that microwave antigen retrieval significantly enhances the immunoreactivity of SNAP-25, chromogranin A, synaptophysin, GAP-43, ubiquitin and tau, in addition to that of beta APP, in formalin-fixed, paraffin-embedded tissue, and reveals NF68 antigenicity where it was not previously detectable.

Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein.

Severe non-missile head injury commonly results in a form of brain damage known as diffuse axonal injury (DAI). The histological diagnosis of DAI is made by silver staining for the presence of axonal retraction balls. This feature takes about 24 h to develop and does not allow for the early histological diagnosis of DAI. We have used immunocytochemistry for the beta-amyloid precursor protein (beta APP) as a marker for axonal injury in formalin-fixed, paraffin-embedded sections of human brain. Axonal beta APP immunoreactivity was present in all cases which had survived for 3 h or more. This was true even where the degree of head injury did not appear to be severe, supporting the theory that DAI is a severe form of a more common phenomenon of axonal injury which occurs after cerebral trauma. beta APP immunoreactivity was also found in some non-head injured cases and so cannot be considered to be a specific marker for trauma. The results show that beta APP immunocytochemistry may be useful in the detection of traumatic axonal injury in its early stages, before the formation of axonal retraction balls, provided care is taken to exclude other causes of such immunoreactivity.

Distribution of choline acetyltransferase immunoreactive axons and terminals in the rat and ferret brainstem.

A survey was made of the density of the cholinergic innervation of different parts of the brainstem of the rat and ferret. Sections of rat and ferret brainstems were stained for choline acetyltransferase (ChAT) immunoreactivity by using a sensitive immunocytochemical method. Adjacent sections were stained for acetylcholinesterase activity or Nissl substance. The density of the distribution of fine calibre, varicose ChAT-positive axons, assumed to represent cholinergic terminals, was categorised arbitrarily into high, medium, or low. A high density of ChAT-positive terminals was found in all or parts of these structures: interpeduncular nucleus, superficial grey layer of the superior colliculus (ferret), intermediate layers of the superior colliculus, lateral part of the central grey (rat), an area medial to the parabigeminal nucleus (rat), pontine nuclei, ventral tegmental nucleus (rat), midline pontine reticular formation, and an area ventral to the exit point of the 5th nerve (ferret). A medium density of ChAT-positive terminals was observed in all or parts of: the substantia nigra zona compacta (ferret), ventral tegmental area (ferret), superficial grey layer of the superior colliculus, intermediate and deep layers of the superior colliculus, lateral central grey, area medial to the parabigeminal nucleus, inferior colliculus, dorsal tegmental nucleus, ventral tegmental nucleus (ferret), pontine nuclei, ventral nucleus of the lateral lemniscus (ferret), midline pontine reticular formation, ventral cochlear nucleus, dorsal cochlear nucleus, lateral superior olive, spinal trigeminal nuclei, prepositus hypoglossal nucleus, lateral reticular nucleus, paragigantocellular nucleus, and the dorsal column nuclei including the cuneate, external cuneate, and gracile nuclei. A low density of ChAT-positive terminals was seen throughout the remainder of the brainstem of the rat and ferret, but these terminals were absent from the medial superior olive, substantia nigra zona reticulata (rat), and the central part of the ferret lateral superior olive. A pericellular-like distribution of ChAT-positive terminals was observed in the ventral cochlear nucleus and in association with some of the cells of the nucleus of the mesencephalic tract of the trigeminal nerve. A climbing fibre type arrangement of ChAT-positive terminals was found in the substantia nigra zona compacta (ferret) and medial reticular formation. In general, the distribution of staining for AChE activity reflected that of the distribution of ChAT immunoreactivity in the brainstem, except in a few regions where there were also species differences in the distribution of ChAT-positive terminals, e.g., in the superficial grey layer of the superior colliculus and in the substantia nigra.(ABSTRACT TRUNCATED AT 400 WORDS)

Organisation of the visceral solitary tract nucleus in the ferret as defined by the distribution of choline acetyltransferase and nerve growth factor receptor immunoreactivity.

The cholinergic innervation of the visceral component of the nucleus of the solitary tract in the ferret was investigated by using choline acetyltransferase immunocytochemistry. The subdivisions of the ferret solitary tract nucleus as defined by Nissl architectonics were found to correspond to most of those previously assigned to the cat solitary tract nucleus. The subnuclei of the ferret solitary tract nucleus were also outlined by using immunohistochemical and histochemical methods to stain for nerve growth factor (NGF) receptor and acetylcholinesterase, respectively. In particular, the gelatinosus and interstitial subnuclei stain intensely for NGF receptor immunoreactivity and for acetylcholinesterase activity. Since abundant NGF receptor immunoreactivity is observed also in the nodose ganglion and in the solitary tract, it was assumed that the gelatinosus and the interstitial subnuclei represent the principal sites of termination of primary visceral afferents. A rich choline acetyltransferase-positive terminal axonal arborization was located in all of the subdivisions of the solitary tract nucleus but was found to be lacking in the gelatinosus and interstitial subnuclei. A small number of giant choline acetyltransferase-positive axon terminals was seen in the subnucleus gelatinosus but was assumed to be of doubtful functional significance because these terminals derive from only one or two large axons on each side of the brain. The weak cholinergic innervation of the gelatinosus and interstitial subnuclei and the stronger innervation of the other subnuclei suggest that acetylcholine has a more important role in the secondary rather than the primary processing of afferent visceral information. Because the distribution of acetylcholinesterase activity in the nucleus of the solitary tract matches that of the NGF receptor immunoreactivity rather than that of the cholinergic acetyltransferase immunoreactivity, a non-cholinergic function for acetylcholinesterase may dominate in the solitary tract nucleus of the ferret.

Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat.

Serotonergic and catecholaminergic neurons are known to project from the brainstem to the spinal cord. However, evidence for a bulbo-spinal projection that is cholinergic is sparse despite immunocytochemical and physiological evidence for a cholinergic influence on the cord. In this study we examined the possibility of a direct cholinergic bulbo-spinal projection in the rat using a combination of retrograde axonal tracing techniques and choline acetyltransferase immunocytochemistry. Although many cells were found to project to the cord from the brainstem, none were identified as being cholinergic, confirming previous evidence that the cholinergic innervation of the cord is intrinsic.

LH responses of female naked mole-rats, Heterocephalus glaber, to single and multiple doses of exogenous GnRH.

To investigate possible differential pituitary secretion of LH in breeding and non-breeding female naked mole-rats, the LH responses to administration of exogenous GnRH were measured in 55 females from 20 captive colonies. Single doses of 0.1, 0.5 or 1.0 micrograms GnRH produced a significant rise in plasma LH concentrations 20 min after s.c. injection in breeding and non-breeding females at all doses (P less than 0.001). While at the highest dose of 1.0 microgram there was no difference in the LH response between breeding and non-breeding females, as the dose was lowered there was a progressive decline in the LH response in non-breeding females such that, at the 0.1 microgram dose, GnRH produced only a small, but significant, increase in plasma LH (1.3 +/- 0.2 to 2.9 +/- 0.5 mi.u./ml, N = 5) compared with breeding females (3.4 +/- 0.8 to 9.6 +/- 2.0 mi.u./ml, N = 6). The LH responses of the latter were not significantly reduced at the lower doses of GnRH. The apparent lack of sensitivity to low doses of exogenous GnRH in non-breeding females was reversed by 4 consecutive 1-h injections of 0.1 microgram, which produced a rise in LH from 1.2 +/- 0.2 to 9.0 +/- 0.2 mi.u./ml (N = 4), comparable to that of breeding females given a single injection of 0.1 microgram GnRH. These results suggest that the anterior pituitary in non-breeding female naked mole-rats is less sensitive to low doses of exogenous GnRH than in breeding females, possibly due to a lack of priming by endogenous GnRH. Therefore, the socially-induced block to ovulation in non-breeding female naked mole-rats may be due to inhibition of hypothalamic GnRH secretion.