Acetylcholinesterase Histochemistry Research Articles

Basal forebrain cholinergic cell attachment and neurite outgrowth on organotypic slice cultures of hippocampal formation

Distributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1–21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective fluorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture. These studies demonstrate a non-random pattern of attachment or differentiation of basal forebrain cholinergic neurons when these cells are seeded onto cultured cortical slices; this pattern mimics the normal patterns of basal forebrain cholinergic projections to these cortical regions. These data suggest that the factors that normally guide basal forebrain-derived cholinergic axons to their target cells in vivo are present and detectable in this model system.

Rectal biopsy: what is the optimal procedure?

Rectal suction biopsy (RSB) is a well-known diagnostic procedure for disorders of bowel motility such as Hirschsprung's disease (HD). However, there are few reports about the optimal method of obtaining rectal tissue. We introduce a new technique using Gruenwald's nasal cutting forceps (NCF). From 1986 to 1999, we performed 130 sets of rectal biopsies in patients suspected of having HD. In group I (1986 to 1994), 68 sets of three-site biopsies (2, 3, and 5 cm above the dentate line) were performed using a conventional blind RSB technique. In group II (1995 to 1999), 62 sets of one-site biopsies (2 cm above the dentate line) were performed using Gruenwald's NCF after anal dilatation during general anesthesia. Hematoxylin-eosin staining and acetylcholinesterase histochemistry were used to examine all specimens. Biopsy specimens in group II (4.39 +/- 1.07 mm(2)) were larger than in group I (1.59 +/- 0.39 mm(2)) ( P < 0.01). In 18 cases (26 %) in group I, normal and HD bowel could not be differentiated because the specimens were too small to detect ganglion cells (i.e., only lamina propria [9 cases] or a small area of submucosa [9 cases] was present). These cases required repeat biopsy. All cases of HD diagnosed in group I (n = 20) were based on the findings of biopsies taken at 2 cm; biopsies from 3 and 5 cm did not provide additional information. There were 2 cases of post-biopsy hemorrhage in group I. In group II, 18 subjects were diagnosed with HD and 39 were confirmed to have normal bowel. There were no complications and repeating the biopsy was unnecessary. Three cases of hypoganglionosis (1 in group I and 2 in group II) were missed because the myenteric plexus abnormalities could not be detected by RSB. Intestinal neuronal dysplasia (IND) was diagnosed in 5 cases (2 in group I by repeat full-thickness biopsy and 3 in group II by rectal biopsy). We conclude that our new technique is advantageous and safe to differentiate between normal bowel, HD, and even IND on the basis of a single biopsy taken 2 cm above the dentate line. The biopsy can be taken under direct vision and is histopathologically accurate.

On the intrinsic innervation of the epididymis of the camel (Camelus dromedarius)

The innervation of the camel epididymis was studied in 26 apparently healthy, sexually mature animals aged between 4 and 12 years. The material was collected during the different seasons of the year. Generally, five samples were taken from each epididymis. To demonstrate the general innervation pattern, immunohistochemical reactions to protein gene product-9.5, neurofilaments and neuron-specific enolase were used, in addition to acetylcholinesterase histochemistry. The nerve supply of the epididymis comes from two sources: (1) The majority of fibers come from the N. spermaticus inferior and accompany the deferent duct. (2) Another contribution stems from the N. spermaticus superior and enters the head region of the epididymis. From the exterior, the nerves penetrate the capsule of the organ to reach the interductular connective tissue. The terminal ramifications are observed directly within the wall of the duct and the wall of the epididymal arteries. The veins of the camel epididymis are not innervated. In the wall of the ductus epididymidis, the nerve fibers form plexuses at the subepithelial level and in the muscular coat. The amount of nerve fibers increases from the head to the tail, paralleling an increase in the intrinsic musculature. The intramural and interductular innervation of epididymal body and tail shows clear seasonal variations: More fibers and stronger reactions are observed during the winter season; the lowest density and the weakest reactions occur during the summer season. All epididymal nerves of the camel are unmyelinated. The majority of the intramural fibers and all in the arterial wall represent postjunctional sympathetic axons, but in the intramural plexuses of the duct a considerable number of cholinergic fibers are also present. Neuropeptide Y is the most frequent peptidergic transmitter and generally co-localized with dopamine-beta-hydroxylase in the sympathetic axons. Vasoactive intestinal polypeptide has a distribution similar to that of the cholinergic fibers. Calcitonin gene-related peptide-positive axons occur in moderate numbers, but never in the arterial innervation. Together with the relatively rare substance P-containing fibers, the calcitonin gene-related peptide-positive axons seem to represent the only sensory nerves in the camel epididymis.

Demonstration of nitrergic and cholinergic innervation in whole-mount preparations of rabbit, pig, and human upper urinary tract.

To investigate the distribution of nitrergic and cholinergic innervation in rabbit, pig, and human upper urinary tract, (UUT) whole-mount preparations and frozen sections were stained with nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and acetylcholinesterase (AChE) histochemistry. NADPH-diaphorase and AChE staining demonstrated two neuronal plexuses in the submucous and muscular layer of the UUT in all three species. The presence of nitrergic and cholinergic neuronal networks in the normal UUT suggests that autonomic innervation may play an important role in the transmission of ureteral peristalsis.

Magnetic stimulation accelerating rehabilitation of peripheral nerve injury.

The effect of magnetic stimulation (MS) on sciatic nerve injury was observed. After sciatic nerve was crushed in 40 Sprague Dawley (SD) rats, one randomly selected group (group D) was subjected, from the 4th day post-operatively to 3 min of continuous 70% of maximum output of MS daily for 8 weeks. The other group (group E) served as a control group. The nerve regeneration and motor function recovery were evaluated by walking track analysis (sciatic function index, SFI; toe spreading reflex, TSR), electrophysiological, histological and acetylcholineesterase histochemistry. The SFI in the group D was greater than in the group E with the difference being statistically significant (P < 0.01). TSR reached its peak on the 4th day in the group D and on the 10th day in the group E respectively. The amplitude and velocity of MCAP and NCAP in the group D was greater than in the group E with the difference being statistically significant (P < 0.01), while the latency and duration of MCAP and NCAP in the group D were less than in the group E with the difference being also statistically significant (P < 0.01). Histological examination showed the mean axon count above the lesion for thick myelinated fibers (> 6.5 microns) in the group D was greater than in the control group with the difference being statistically significant (P < 0.01), while the mean axon count below the lesion for thick myelinated fibers was less than that in the group E with the difference being statistically significant (P < 0.01). The mean axon count above the lesion for thin myelinated fibers (2-6.5 microns) in the group D was greater than that in the group E with the difference being statistically significant (P < 0.05), while the mean axon count below the lesion for thin myelinated in the group D was greater than that in the group E with the difference being statistically significant (P < 0.01). Acetylcholine esterase examination showed that the MS could significantly increase the number of the motor neurons. There was no significant difference in the number of the motor neurons between the treatment side and the normal side (P > 0.05). It can be concluded that MS can enhance functional recovery and has a considerable effect in the treatment of the peripheral nerve injury.

Effects of infantile/prepubertal chronic estrogen treatment and chemical sympathectomy with guanethidine on developing cholinergic nerves of the rat uterus.

The innervation of the uterus is remarkable in that it exhibits physiological changes in response to altered levels in the circulating levels of sex hormones. Previous studies by our group showed that chronic administration of estrogen to rats during the infantile/prepubertal period provoked, at 28 days of age, an almost complete loss of norepinephrine-labeled sympathetic nerves, similar to that observed in late pregnancy. It is not known, however, whether early exposure to estrogen affects uterine cholinergic nerves. Similarly, it is not known to what extent development and estrogen-induced responses in the uterine cholinergic innervation are affected by the absence of sympathetic nerves. To address this question, in this study we analyzed the effects of infantile/prepubertal chronic estrogen treatment, chronic chemical sympathectomy with guanethidine, and combined sympathectomy and chronic estrogen treatment on developing cholinergic nerves of the rat uterus. Cholinergic nerves were visualized using a combination of acetylcholinesterase histochemistry and the immunohistochemical demonstration of the vesicular acetylcholine transporter (VAChT). After chronic estrogen treatment, a well-developed plexus of cholinergic nerves was observed in the uterus. Quantitative studies showed that chronic exposure to estrogen induced contrasting responses in uterine cholinergic nerves, increasing the density of large and medium-sized nerve bundles and reducing the intercept density of fine fibers providing myometrial and perivascular innervation. Estrogen-induced changes in the uterine cholinergic innervation did not appear to result from the absence/impairment of sympathetic nerves, because sympathectomy did not mimic the effects produced by estrogen. Estrogen-induced responses in parasympathetic nerves are discussed, considering the direct effects of estrogen on neurons and on changes in neuron-target interactions.

Low levels of estrogen significantly diminish axonal sprouting after entorhinal cortex lesions in the mouse.

This study tested the hypothesis that estrogen enhances axonal sprouting in the hippocampal formation in the female mouse. The entorhinal cortex was unilaterally lesioned with ibotenic acid in control mice and in ovariectomized mice that were treated with a high dose of, a moderate dose of, or zero estrogen supplementation pellets. Four weeks later the density of staining for synaptophysin immunoreactivity and acetylcholinesterase (AChE) histochemistry was measured in the molecular layer of the dentate gyrus. In control mice, lesions of the lateral part of the entorhinal cortex increased synaptophysin and acetylcholinesterase staining (i.e., indicative of axonal sprouting) in the outer one-third of the molecular layer of the dentate gyrus. Mice receiving high and moderate estrogen supplementation displayed the same sprouting response; however, in ovariectomized mice the sprouting response was significantly reduced (to nearly nothing). Thus, in ovariectomized compared with control mice the lesion-induced sprouting response is severely blunted, and this effect is reversed by estrogen supplementation. Together, these findings suggest that estrogen plays a prominent role in promoting neuronal plasticity and remodeling in the dentate gyrus.

Low Levels of Estrogen Significantly Diminish Axonal Sprouting after Entorhinal Cortex Lesions in the Mouse

This study tested the hypothesis that estrogen enhances axonal sprouting in the hippocampal formation in the female mouse. The entorhinal cortex was unilaterally lesioned with ibotenic acid in control mice and in ovariectomized mice that were treated with a high dose of, a moderate dose of, or zero estrogen supplementation pellets. Four weeks later the density of staining for synaptophysin immunoreactivity and acetylcholinesterase (AChE) histochemistry was measured in the molecular layer of the dentate gyrus. In control mice, lesions of the lateral part of the entorhinal cortex increased synaptophysin and acetylcholinesterase staining (i.e., indicative of axonal sprouting) in the outer one-third of the molecular layer of the dentate gyrus. Mice receiving high and moderate estrogen supplementation displayed the same sprouting response; however, in ovariectomized mice the sprouting response was significantly reduced (to nearly nothing). Thus, in ovariectomized compared with control mice the lesion-induced sprouting response is severely blunted, and this effect is reversed by estrogen supplementation. Together, these findings suggest that estrogen plays a prominent role in promoting neuronal plasticity and remodeling in the dentate gyrus.

Effects of 192 IgG-saporin on acetylcholinesterase histochemistry in male and female rats

Sex hormones may exert neuroprotective effects in various models of brain lesions. Male and female Long–Evans rats were subjected to intracerebroventricular injections of 2 μg 192 IgG-saporin or vehicle. Starting 2 days before surgery, half the male rats were treated with estradiol for 7 days. Three weeks after surgery, they were sacrificed for histochemical staining of acetylcholinesterase (AChE) and densitometric evaluations. The lesion induced a substantial to dramatic decrease of the AChE-positive fiber density in the cingulate, somatosensory, piriform, retrosplenial and perirhinal cortices, and in the hippocampus. Weak effects were found in the striatum. There was no significant decrease in the dorsal thalamus. Sex had no significant effect on AChE-positive staining in any brain area. In males, estradiol treatment did not alter the effects of 192 IgG-saporin. These results show that sex or estradiol treatment in male rats does not interfere with the immunotoxic effects of intracerebroventricular injections of 192 IgG-saporin on cholinergic projections from the basal forebrain.

Selective chemical ablation of the enteric plexus in mice.

Although genetically aganglionic mice such as piebald lethal and lethal spotted mice exhibit striking similarities to the human condition of Hirschsprung's disease (HD), the aganglionic segment is very short and always located in the distal part of the rectum. Topical application of benzalkonium chloride (BAC) to the rectum of rats has been reported to result in segmental aganglionosis. To induce chemical ablation of the enteric plexus in mice to produce an aperistaltic narrow segment simulating HD, 32 mice were divided into three groups: (1) abdominal (n=12), for sigmoid colon treatment; (2) rectal (n=10), for rectum treatment; and (3) controls (n=10). For groups 1 and 2, 0.1% BAC was applied to a 1-cm serosal surface of the bowel for 15 min. In the controls, isotonic saline was applied in this fashion. A detailed histologic examination was performed using hematoxylin and eosin staining and acetylcholinesterase histochemistry. Ten animals (9 in group 1 and 1 in group 2) died 1 to 9 weeks after BAC treatment. Autopsy revealed a narrow segment of bowel at the site of BAC treatment and marked dilatation of the bowel proximal to the narrow segment. The remaining animals were killed 12 weeks after BAC treatment. Histologic examination demonstrated normal myenteric and submucous plexuses in the controls, whereas there was a total lack of innervation in the BAC-treated segments.Topical application of BAC thus successfully produced a narrow aganglionic segment of bowel in normal mice. This model provides the basis for future studies to investigate the pathophysiology of HD and megacolon and for comparison with genetically aganglionic mice.

Abnormalities of C-Kit–positive cellular network in isolated hypoganglionosis

Background/Purpose: C-Kit-positive interstitial cells of Cajal (ICCs) have a key role in the normal motility function and development of the bowel. They are pacemaker cells, which facilitate active propagation of electrical events and neurotransmission in the bowel wall. ICCs are present in the bowel as myenteric ICCs (ICCmyS) and muscular ICCs (ICCmusS). The aim of this study was to examine the distribution of c-Kit-positive ICCs and their relationship to the autonomic intrinsic innervation in bowel specimens from patients with isolated hypoganglionosis. Methods: Full-thickness large bowel specimens were obtained from 6 patients with hypoganglionosis and from 4 patients during bladder augmentation (controls). Frozen sections and whole-mount preparations were stained using c-Kit immunohistochemistry, nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, and acetylcholinesterase (AChE) histochemistry and evaluated using normal brightfield and confocal laser scanning microscopy. Results: NADPH-diaphorase and AChE histochemistry findings showed characteristic histologic features of hypoganglionosis, eg, sparse and small myenteric ganglia and low or absent AChE activity in the lamina propria. Myenteric plexus in the normal bowel was surrounded by a dense network of c-Kit-positive ICCmyS, whereas in hypoganglionosis sparse isolated ICCmyS were found. C-Kit-positive ICCmusS were reduced markedly in the longitudinal and circular muscle layer and at the innermost part of the circular muscle in hypoganglionosis. Conclusion: Deficient expression of c-Kit-positive myenteric and muscular ICCs in the hypoganglionic colon may contribute to the motility dysfunction in the affected bowel. J Pediatr Surg 37:709-714. Copyright 2002, Elsevier Science (USA). All rights reserved.

Immunohistochemical investigations of the autonomous nerve distribution in the testis of the camel ( Camelus dromedarius)

The distribution of autonomous nerves in the testis of the camel was studied by immunohistochemical methods. A total of 26 testes was collected during the different seasons of the year. As pan-neuronal markers, antibodies to protein gene product 9.5 and to neurofilaments are superior to antibodies against neuron-specific enolase and acetylcholinesterase histochemistry for the description of the nerves in the camel testis. Testicular nerves reach the camel testis by three access-routes as (1) funicular contribution, (2) mesorchial contribution and (3) as caudal contribution. The main target for testicular nerves is the arterial vascular tree of the organ, whereas all veins of testis and pampiniform plexus are devoid of any innervation in the camel. In the wall of the arteries, the nerves form a plexus at the media-adventitia border. The density of the arterial plexuses increases along the vascular tree: smaller septal and mediastinal arteries are better innervated than albugineal arteries and the latter better than the A. testicularis. The nerves in the septula testis, in the mediastinum and between the Leydig cells show clear seasonal changes, being particularly abundant in autumn and particularly scarce in spring. The nerves that reach the camel testis are unmyelinated and represent in the vast majority postjunctional sympathetic neurons. Cholinergic fibers are absent in the camel testis. Neuropeptide Y is the dominating peptidergic transmitter in the testicular nerves and colocalized with noradrenaline in the same axons. Vasoactive intestinal polypeptide-containing fibers reach the camel testis exclusively as parts of the caudal nervous contribution via the ligamentous bridge between testis and epididymal tail and are restricted to the caudal pole of the testis. Calcitonin gene-related peptide-positive axons are not frequent in the camel testis; nevertheless, they seem to be the most important sensory pathway of this organ.

Comparative study of the neuroprotective effect of dehydroepiandrosterone and 17β-estradiol against 1-methyl-4-phenylpyridium toxicity on rat striatum

The effects of dehydroepiandrosterone, estradiol and testosterone on 1-methyl-4-phenylpyridium (MPP +)-induced neurotoxicity of the nigrostriatal dopaminergic system were examined in rat. They were subjected to a unilateral intrastriatal infusion of the following treatment conditions: MPP + alone or co-injection of MPP + plus each hormone. Four days after injection, concentrations of dopamine and their metabolites were determined from the corpus striatum. To corroborate the neurochemical data an immunohistochemical analysis of tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase histochemistry in the striatum was performed. Moreover, we performed a dose–response study of the three hormones on the high-affinity dopamine transport system in rat striatal synaptosomes. Rats co-injected within the striatum with MPP + and either dehydroepiandrosterone or estradiol had significantly greater concentrations of dopamine and less tyrosine hydroxylase-immunoreactive fibers and acetylcholinesterase fiber density loss compared with their respective controls. In addition, 4 days after injection, the brain was fixed and cut into coronal sections, and was immunostained with major histocompatibility complex class II antigens for activated microglia, and glial fibrillary acidic protein for activated astrocytes. Dehydroepiandrosterone also attenuated microglial cell activation. In contrast, testosterone showed reductions in dopamine concentrations similar to those obtained by MPP +. The protective effect of dehydroepiandrosterone against the MPP + neurotoxic dopaminergic system may be produced by its partial prevention of MPP + inhibition of NADH oxidase activity, whereas the estradiol may function as a neuroprotectant by reducing the uptake of MPP + into dopaminergic neurons. Our findings we suggest indicate that dehydroepiandrosterone and estradiol by a non-genomic effect may have an important modulatory action, capable of attenuating degeneration within the striatum, and in this way serve as neuroprotectants of the nigrostriatal dopaminergic system.

Sexual dimorphism in neuromuscular junction size on a muscle used in courtship by green anole lizards

The green anole lizard exhibits seasonal courtship behavior that is sexually dimorphic. This courtship consists of the extension of a bright red throat fan (dewlap) associated with head-bobbing display behavior. While males extend their dewlaps in aggressive encounters as well as in courtship, females use their considerably smaller dewlaps much less frequently and mainly in agonistic encounters. In parallel, a number of components of the neuromuscular system controlling dewlap extension are greater in males than in females during the breeding season, including dewlap motoneuron soma size and muscle fiber size and number. These features do not seem to change substantially in adulthood, despite a dramatic decline in dewlap use during the nonbreeding season. We explored the morphology of this neuromuscular system in more detail in the present experiment in males and females during both the breeding and nonbreeding seasons. Fiber and whole muscle length (approximately perpendicular to the fibers) were measured. Acetylcholinesterase histochemistry was used to visualize neuromuscular junctions (NMJs), and the surface area and density of NMJs were assessed for each animal. During the breeding season, NMJ size was larger in males than in females, but NMJ density along each fiber was equivalent between the sexes. In addition, whole muscle length and that of individual muscle fibers, was larger in males than in females. However, when corrected for body size, the sex difference in muscle fiber length disappeared. In the nonbreeding season, the sexual dimorphisms were maintained, suggesting that these features do not change substantially due to differences in circulating testosterone or a difference in use across seasons. Overall, these results are consistent with the idea that enhanced NMJ size is a relatively stable feature of the dewlap muscle in adulthood that either facilitates or is a consequence of using a larger muscle to extend a bigger dewlap in males compared to females.

Long-term synaptic alteration in the rat hippocampal CA3 field following an entorhinal cortex lesion.

The entorhinal cortex is a key initial relay for cortical input to the hippocampus. To better understand hippocampal dysfunction resulting from early entorhinal cortex involvement in Alzheimer's disease, we stereotaxically injected ibotenic acid to produce unilateral entorhinal cortex lesions in rats. We then serially examined the CA3 hippocampal region by neuronal counts, histochemistry for acetylcholinesterase, and synaptophysin immunohistochemistry. Over 12 months, the neuronal counts did not change. Acetylcholinesterase-positive fibers were persistently but non-progressively beginning at 3 months. Synaptophysin immunoreactivity progressively declined over 12 months. Since much of the entorhinal cortex output proceeds to CA3 via the dentate gyrus, transsynaptic degeneration is suspected.

Short-term consequences of N-methyl- D-aspartate excitotoxicity in rat magnocellular nucleus basalis: effects on in vivo labelling of cholinergic neurons

Cholinergic neurons of the basal forebrain form one of the neuron populations that are susceptible to excitotoxic injury. Whereas neuropharmacological studies have aimed at rescuing cholinergic neurons from acute excitotoxic attacks, the short-term temporal profile of excitotoxic damage to cholinergic nerve cells remains largely elusive. The effects of N-methyl- D-aspartate (NMDA) infusion on cytochemical markers of cholinergic neurons in rat magnocellular nucleus basalis were therefore determined 4, 24 and 48 h post-lesion. Additionally, the influence of excitotoxic damage on the efficacy of in vivo labelling of cholinergic neurons with carbocyanine 3–192IgG was investigated. Carbocyanine 3–192IgG was unilaterally injected in the lateral ventricle. Twenty-four hours later, NMDA (60 nM/μl) was infused in the right magnocellular nucleus basalis, while control lesions were performed contralaterally. Triple immunofluorescence labelling for carbocyanine 3–192IgG, NMDA receptor 2A and B subunits and choline-acetyltransferase (ChAT) was employed to determine temporal changes in NMDA receptor immunoreactivity on cholinergic neurons. The extent of neuronal degeneration was studied by staining with Fluoro-Jade. Moreover, changes in the numbers of ChAT or p75 low-affinity neurotrophin receptor immunoreactive neurons, and the degree of their co-labelling with carbocyanine 3–192IgG were determined in basal forebrain nuclei. The effects of NMDA-induced lesions on cortical projections of cholinergic nucleus basalis neurons were studied by acetylcholinesterase (AChE) histochemistry. Characteristic signs of cellular damage, as indicated by decreased immunoreactivity for NMDA receptors, ChAT and p75 low-affinity neurotrophin receptors, were already detected at the shortest post-lesion interval investigated. Fluoro-Jade at 4 h post-lesion only labelled the core of the excitotoxic lesion. Longer survival led to enhanced Fluoro-Jade staining, and to the decline of ChAT immunoreactivity reaching a maximum 24 h post-surgery. Significant loss of p75 low-affinity neurotrophin receptor immunoreactivity and of cortical AChE-positive projections only became apparent 48 h post-lesion. Carbocyanine 3–192IgG labelling in the ipsilateral basal forebrain exceeded that of the contralateral hemisphere at all time points investigated and progressively declined in the damaged magnocellular nucleus basalis up to 48 h after NMDA infusion. The present study indicates that excitotoxic lesion-induced alteration of cholinergic neuronal markers is a rapid and gradual process reaching its maximum 24 h post-surgery. Furthermore, in vivo labelling of cholinergic neurons may be applied to indicate neuronal survival under pathological conditions, and enable to follow their degeneration process under a variety of experimental conditions.

Heightened seizure severity in somatostatin knockout mice

Patients and experimental models of temporal lobe epilepsy display loss of somatostatinergic neurons in the dentate gyrus. To determine if loss of the peptide somatostatin contributes to epileptic seizures we examined kainate-evoked seizures and kindling in somatostatin knockout mice. Somatostatin knockout mice were not observed to experience spontaneous seizures. Timm staining, acetylcholinesterase histochemistry, and immunocytochemistry for NPY, calbindin, calretinin, and parvalbumin revealed no compensatory changes or developmental abnormalities in the dentate gyrus of somatostatin knockout mice. Optical fractionator counting of Nissl-stained hilar neurons showed similar numbers of neurons in wild type and somatostatin knockout mice. Mice were treated systemically with kainic acid to evoke limbic seizures. Somatostatin knockout mice tended to have a shorter average latency to stage 5 seizures, their average maximal behavioral seizure score was higher, and they tended to be more likely to die than controls. In response to kindling by daily electrical stimulation of the perforant path, to more specifically challenge the dentate gyrus, mean afterdischarge duration in somatostatin knockout mice was slightly longer, but the number of treatments to five stage 4–5 seizures was similar to controls. Although we cannot exclude the possibility of undetected compensatory mechanisms in somatostatin knockout mice, these findings suggest that somatostatin may be mildly anticonvulsant, but its loss alone is unlikely to account for seizures in temporal lobe epilepsy.

Mechanisms of peritumoural brain dysfunction: metabolic and neuroreceptor findings in striatal C6 glioma

The aetiology of the peritumoural brain dysfunction that is rectified by steroids is unknown. To determine potential aspects of its pathophysiological basis we performed metabolic, histochemical and neuroreceptor studies in rodents with striatal C6 gli oma. This model is known to cause focal neurobehavioural and electrophysiological dysfunction. The fully quantitative [14C]-2-deoxyglucose autoradiographic technique of measuring local cerebral metabolism of glucose (LCMRglu) showed raised LCMRglu (22–29%) in the pallidum, substantia nigra and endopeduncular nucleus. Acetylcholinesterase (AChE) histochemistry and a range of ligand binding studies for dopamine type 1 and 2, and serotonergic 5-HT2receptors were negative in the tumour and normal in peritumoural brain. 5-HT uptake sites and strong peripheral benzodiazepine receptor expression were present in the tumour. There was extensive up-regulation of peripheral benzodiazepine receptor expression in the peritumoural brain. These studies show there is metabolic dysregulation in brain regions functionally connected to, but anatomically distant from the striatum. There is also a peritumoural region of up-regulated receptors that have many, predominantly inhibitory, functions. The relationship of these findings to peritumoural brain dysfunction is discussed.

Presence and distribution of sensory nerve fibers in human peritoneal adhesions.

To assess the distribution and type of nerve fibers present in human peritoneal adhesions and to relate data on location and size of nerves with estimated age and with clinical parameters such as reports of chronic pelvic pain. Peritoneal adhesions are implicated in the cause of chronic abdominopelvic pain, and many patients are relieved of their symptoms after adhesiolysis. Adhesions are thought to cause pain indirectly by restricting organ motion, thus stretching and pulling smooth muscle of adjacent viscera or the abdominal wall. However, in mapping studies using microlaparoscopic techniques, 80% of patients with pelvic adhesions reported tenderness when these structures were probed, an observation suggesting that adhesions themselves are capable of generating pain stimuli. Human peritoneal adhesions were collected from 25 patients undergoing laparotomy, 20 of whom reported chronic pelvic pain. Tissue samples were prepared for histologic, immunohistochemical, and ultrastructural analysis. Nerve fibers were characterized using antibodies against several neuronal markers, including those expressed by sensory nerve fibers. In addition, the distribution of nerve fibers, their orientation, and their association with blood vessels were investigated by acetylcholinesterase histochemistry and dual immunolocalization. Nerve fibers, identified histologically, ultrastructurally, and immunohistochemically, were present in all the peritoneal adhesions examined. The location of the adhesion, its size, and its estimated age did not influence the type of nerve fibers found. Further, fibers expressing the sensory neuronal markers calcitonin gene-related protein and substance P were present in all adhesions irrespective of reports of chronic abdominopelvic pain. The nerves comprised both myelinated and nonmyelinated axons and were often, but not invariably, associated with blood vessels. This study provides the first direct evidence for the presence of sensory nerve fibers in human peritoneal adhesions, suggesting that these structures may be capable of conducting pain after appropriate stimulation.

Postnatal development of cholinergic system in mouse basal forebrain: acetylcholinesterase histochemistry and choline-acetyltransferase immunoreactivity

The distribution of acetylcholinesterase histochemistry and choline-O-acetyltransferase immunohistochemistry in the basal forebrain was studied in newborn mice (P0) and until 60 days of postnatal life (P60). A weak acetylcholinesterase activity was found at P0 and P2 in the anterior and intermediate parts of the basal forebrain, and higher in the posterior region. The intensity of labeling, neuronal size and dendritic growth seems to increase progressively in all regions of basal forebrain from P4 to P10. The AChE+ cell count shows that in the anterior portion of the magnocellular basal nucleus the number of cells does not vary significantly from birth to the second month of postnatal life. However, in the intermediate and posterior portions of the nucleus the mean number of labeled cells increases significantly from birth to the end of the second week of postnatal life (P13). The choline-acetyltransferase immunoreactivity appears only detectable at the end of the first week (P6) as a slight immunoreaction, which increases progressively in intensity at P8, and at P10 seems to attain the same intensity of labeling found at P60. These results seem to indicate that the acetylcholinesterase could have a non-classic cholinergic role in the first stages of postnatal development, acting as a growth and cellular differentiation factor.