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Background and Objectives: To describe the etiology and clinical course of pediatric acute-onset unilateral peripheral facial palsy (FP), to define factors that distinguish Bell's palsy from Lyme-related FP (LRFP), and to determine if early corticosteroid use impacts facial strength recovery in Bell's palsy or LRFP., Methods: Retrospective cohort study of children 1 to 18 years old who received clinical care within our pediatric clinical care network (Lyme-endemic region) between 2013 and 2018 for acute-onset unilateral peripheral FP., Results: The study included 306 children; 82 (27%) had LRFP, 209 (68%) had Bell's palsy, and 15 (5%) had FP of different etiology. Most children with LRFP presented between June and November (93%), and compared with Bell's palsy, more often had a preceding systemic prodrome, including fever, malaise, headache, myalgias, and/or arthralgias (55% vs 6%, P < .001). Neuroimaging and lumbar puncture did not add diagnostic value in isolated FP. Of the 226 children with Bell's palsy or LRFP with documented follow-up, FP was resolved in all but 1. There was no association between ultimate parent/clinician assessment of recovery and early corticosteroid use., Conclusions: Bell's palsy and LRFP were common causes of pediatric FP in our Lyme endemic region. Systemic prodrome and calendar month may help distinguish LRFP from Bell's palsy at FP onset, guiding antibiotic use. Early corticosteroid use did not impact our measures of recovery, although subtle abnormalities may not have been appreciated, and time to recovery could not be assessed. Future prospective studies using standardized assessment tools at regular follow-up intervals are necessary., (Copyright © 2022 by the American Academy of Pediatrics.)

Objectives: To define the incidence and characteristics of influenza-associated neurologic complications in a cohort of children hospitalized at a tertiary care pediatric hospital with laboratory-confirmed influenza and to identify associated clinical, epidemiologic, and virologic factors., Study Design: This was an historical cohort study of children aged 0.5-18.0 years old hospitalized between 2010 and 2017 with laboratory-confirmed influenza. Children with immune compromise or a positive test due to recent receipt of live virus vaccine or recently resolved illness were excluded. Influenza-associated neurologic complications were defined as new-onset neurologic signs/symptoms during acute influenza illness without another clear etiology., Results: At least 1 influenza-associated neurologic complication was identified in 10.8% (95% CI 9.1-12.6%, n = 131 of 1217) of hospitalizations with laboratory-confirmed influenza. Seizures (n = 97) and encephalopathy (n = 44) were the most commonly identified influenza-associated neurologic complications, although an additional 20 hospitalizations had other influenza-associated neurologic complications. Hospitalizations with influenza-associated neurologic complications were similar in age and influenza type (A/B) to those without. Children with a pre-existing neurologic diagnosis (n = 326) had a greater proportion of influenza-associated neurologic complications compared with those without (22.7% vs 6.4%, P < .001). Presence of a pre-existing neurologic diagnosis (aOR 4.6, P < .001), lack of seasonal influenza vaccination (aOR 1.6, P = .020), and age ≤5 years (aOR 1.6, P = .017) were independently associated with influenza-associated neurologic complications., Conclusions: Influenza-associated neurologic complications are common in children hospitalized with influenza, particularly those with pre-existing neurologic diagnoses. A better understanding of the epidemiology and factors associated with influenza-associated neurologic complications will direct future investigation into potential neuropathologic mechanisms and mitigating strategies. Vaccination is recommended and may help prevent influenza-associated neurologic complications in children., (Copyright © 2021. Published by Elsevier Inc.)

Objectives: Transmasculine individuals, those assigned female sex at birth but who identify as masculine, have high rates of suicidal behavior and often suffer from chest dysphoria (discomfort and distress from unwanted breast development). Growing numbers of transmasculine youth are pursuing definitive treatment with masculinizing chest surgery (MCS), and adult studies reveal marked benefits of MCS, although little is known about the impact of chest dysphoria on transmasculine youth or the optimal timing of MCS. In this study, we aimed to explore youth experiences of chest dysphoria and the impact of MCS., Methods: Transmasculine youth aged 13 to 21 were recruited from a pediatric hospital-based gender clinic. Participants completed a semistructured qualitative interview exploring the experience of chest dysphoria and thoughts about or experiences with MCS. Interview transcripts were coded by 3 investigators employing modified grounded theory, with the median interrater reliability at κ = 0.92., Results: Subjects ( N = 30) were a mean age of 17.5 years, and 47% had undergone MCS. Youth reported that chest dysphoria triggered strong negative emotions and suicidal ideation, caused a myriad of functional limitations, and was inadequately relieved by testosterone therapy alone. All post-MCS youth reported near or total resolution of chest dysphoria, lack of regret, and improved quality of life and functioning., Conclusions: We observed consensus that chest dysphoria is a major source of distress and can be functionally disabling to transmasculine youth. MCS performed during adolescence, including before age 18, can alleviate suffering and improve functioning. Additional research is needed to develop patient-reported outcome measures to assess the impact of chest dysphoria and MCS., Competing Interests: POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Environmental DNA (eDNA) applications are transforming the standard of characterizing aquatic biodiversity via the presence, location and abundance of DNA collected from environmental samples. As eDNA studies use DNA fragments as a proxy for the presence of organisms, the ecological properties of the complex and dynamic environments from which eDNA is sampled need to be considered for accurate biological interpretation. In this review, we discuss the role that differing environments play on the major processes that eDNA undergoes between organism and collection, including shedding, decay and transport. We focus on a mechanistic understanding of these processes and highlight how decay and transport models are being developed towards more accurate and robust predictions of the fate of eDNA. We conclude with five recommendations for eDNA researchers and practitioners, to advance current best practices, as well as to support a future model of eDNA spatio-temporal persistence.

Aims: To investigate the histological, immunohistochemical and ultrastructural features of the posterior hyaloid membrane (PHM) in its naturally separated state in patients without previous surgery and slit-lamp documentation of antemortem posterior vitreous detachment (PVD)., Methods: A prospective study was commenced in 1992 to recruit patients with physiological PVD from an unselected group of general medical inpatients and ascertain the prevalence of PVD. Postmortem specimens subsequently available were studied to analyse the clinicopathological correlation and processed using standard techniques for histology, immunohistochemistry and electron microscopy., Results: Eighty-five patients were examined with ages ranging from 68 to 98 yrs (mean 83.4 yrs). The posterior hyaloid membrane had clearly separated from the retina in 66% of eyes. Twenty-nine eyes from 15 patients were subsequently studied pathologically. The posterior hyaloid membrane exhibited a uniform cellular component, most densely populated around the Weiss' ring. The cells were characterised by oval or round nuclei, indistinct cytoplasm and were only seen within, or abutting, the weakly eosinophilic posterior hyaloid membrane. The posterior aspect of the posterior hyaloid membrane showed a convoluted appearance staining lightly with haematoxylin and eosin. The detached posterior hyaloid membrane exhibited focal positivity for GFAP and type IV collagen. Electron microscopy demonstrates both fibres and basement membrane associated with the cellular component including hemi-desmosome attachment plaques between the cells and basement membrane., Conclusions: This study illustrates some of the structural differences between the posterior hyaloid membrane and the cortical vitreous gel it envelopes and demonstrates the presence of cells intimately associated with the posterior hyaloid membrane in its naturally separated state. We propose the cellular population integral to the PHM to be designated as laminocytes in order to emphasise their type IV collagen/basement membrane association and planar array within the membrane which separates at posterior vitreous detachment.

A polyclonal antibody raised against a 17 amino acid polypeptide (the predicted C-terminal sequence of the cloned Drosophila melanogaster gamma-aminobutyric acid (GABA) receptor subunit, RDL) was used to investigate the distribution of GABA receptor subunit(s) of this type in the nervous system of the cockroach Periplaneta americana. Intense staining was detected in the calyces of the mushroom bodies, glomeruli of the antennal lobes, lower central body, the corpora cardiaca and several cell layers of the medulla and the lobula regions of the optic lobe. The most intense immunocytochemical staining was in the suboesophageal ganglion. Control sections pre-incubated with the primary antibody and conjugated peptide were not stained. Thus, it appears that a GABA receptor subunit of the RDL type is located in cockroach brain regions involved in the processing of visual, olfactory and mechanosensory inputs to the nervous system. Since the corpora cardiaca reacted to this antiserum, this type of GABA receptor may also be involved in the regulation of neurosecretory activity.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders. Recently mutations have been found in the genes for type V collagen in a small number of people with the most common forms of EDS, types I and II. Here we characterise a COL5A2 mutation in an EDS II family. Cultured dermal fibroblasts obtained from an affected subject synthesised abnormal type V collagen. Haplotype analysis excluded COL5A1 but was concordant with COL5A2 as the disease locus. The entire open reading frame of the COL5A2 cDNA was directly sequenced and a single base mutation detected. It substituted a glycine residue within the triple helical domain (G934R) of alpha2(V) collagen, typical of the dominant negative changes in other collagens, which cause various other inherited connective tissue disorders. All three affected family members possessed the single base change, which was absent in 50 normal chromosomes.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective-tissue disorders characterized by skin fragility, joint laxity, and skeletal deformities. Type V collagen appears to have a causal role in EDS types I and II, which show phenotypic overlap and may sometimes be allelic. Type V collagen can exist as a heterotrimer, [alpha1(V)]2alpha2(V), and it both coassembles with and regulates type I collagen-fibril diameter. Using an intragenic COL5A1 polymorphism, we have demonstrated linkage, at zero recombination, to the same allele in two large British EDS type II families (LOD scores 4.1 and 4.3). Affected members from each family were heterozygous for a point mutation in intron 32 (IVS32:T-25G), causing the 45-bp exon 33 to be lost from the mRNA in approximately 60% of transcripts from the mutant gene. This mutation lies only 2 bp upstream of a highly conserved adenosine in the consensus branch-site sequence, which is required for lariat formation. Although both families shared the same marker allele, we have been unable to identify a common genealogy. This is the first description of a mutation at the lariat branch site, which plays a pivotal role in the splicing mechanism, in a collagen gene. Very probably, the resulting in-frame exon skip has a dominant-negative effect due to incorporation of the mutant proalpha chain into the triple-helical molecule. These findings further confirm the importance of type V collagen in the causation of EDS type II, and the novel collagen mutation indicates the importance of the lariat branch site in splicing.

We describe a 16-year-old patient with Ehlers-Danlos syndrome (EDS) type I and recurrent urinary retention caused by giant bladder diverticulum and review the literature on this association.

Background: Several investigators have demonstrated that routine nasogastric decompression after abdominal surgery is unnecessary and can be safely eliminated, and 1 recent study demonstrated the safety of early oral feedings., Objective: To test the hypothesis that successful early feeding would lead to a shorter duration of hospitalization and, therefore, would be more cost-effective., Patients: Fifty-eight patients with elective colorectal surgery., Methods: Patients were prospectively randomized to 1 of 2 postoperative treatment arms: early feeding (EF group, n = 29) and traditional feeding (TF group, n = 29). All patients in the EF group began a liquid diet on the first postoperative day and were advanced to a regular diet when they consumed 1000 mL in 24 hours. All patients in the TF group began a liquid diet after resolution of the postoperative ileus and were advanced to a regular diet after consuming 1000 mL in 24 hours. Patients were dismissed after tolerating two thirds of the regular diet. Both groups had intraoperative orogastric tubes that were removed at the end of surgery. Nasogastric tubes were inserted for persistent postoperative vomiting., Results: No significant differences were noted in age, types of procedures, or in prior abdominal surgery in either group. No significant differences were seen in rates of nausea (55% in EF vs 50% in TF group) or vomiting (48% in EF vs 33% in TF group). One patient in the EF group had aspiration pneumonia, and anastomotic leak resulted in sepsis and eventual death of 1 patient in the TF group. No significant difference was observed in length of hospital stay between the 2 groups (mean +/- SD, 7.2 +/- 3.3 days in EF vs 8.1 +/- 2.3 days in TF group)., Conclusions: Early oral feeding after elective colorectal surgery is safe. Most of the patients tolerated EF; however, there was no significant difference in duration of hospitalization in these patients.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterised by skin hyperextensibility, joint hypermobility, easy bruising, and cutaneous fragility. Nine discrete clinical subtypes have been classified. We have investigated the molecular defect in a patient with clinical features of Ehlers-Danlos syndromes types I/II and VII. Electron microscopy of skin tissue indicated abnormal collagen fibrillogenesis with longitudinal sections showing a marked disruption of fibril packing giving very irregular outlines to transverse sections. Analysis of the collagens produced by cultured fibroblasts showed that the type V collagen had a population of alpha 1 (V) chains shorter than normal. Peptide mapping suggested a deletion within the triple helical domain. RTPCR amplification of mRNA covering the whole of this domain of COL5A1 showed a deletion of 54 bp. Although six Gly-X-Y triplets were lost, the essential triplet amino acid sequence and C-propeptide structure were maintained allowing mutant protein chains to be incorporated into triple helices. Genomic DNA analysis identified a de novo G+3-->T transversion in a 5' splice site of one COL5A1 allele. This mutation is analogous to mutations causing exon skipping in the major collagen genes, COL1A1, COL1A2, and COL3A1, identified in several cases of osteogenesis imperfecta and EDS type IV. These observations support the hypothesis that type V, although quantitatively a minor collagen, has a critical role in the formation of the fibrillar collagen matrix.

Background: Stereotactic breast biopsy has been developed as a less invasive means of performing biopsy for mammographic abnormalities., Methods: From July 1994 through June 1995, 103 women with mammographic abnormalities requiring biopsy were prospectively evaluated., Results: Fifty-one women had open biopsy, and 52 women had stereotactic biopsy. The average age in both groups was 60 years. Pathology revealed malignancy in 12% of stereotactic biopsies and 13% of open biopsies. Complications occurred in 6% of the open biopsies and 4% of the stereotactic biopsies and were limited to hematomas or seromas. The average cost was $2400 for open biopsy and $650 for stereotactic biopsy (P < 0.01). One hundred and one patients returned for a follow-up mammogram within 6 months, and 1 patient in each group required a second biopsy, which revealed benign pathology. A Patient Satisfaction Survey revealed no significant differences in patient satisfaction between the two types of procedures., Conclusion: There were no differences between open and stereotactic biopsies in regards to diagnostic accuracy, complications, or patient satisfaction. A significant difference was noted in charges during the time frame of our study.

An antibody raised against a peptide based on the C-terminal derived amino acid sequence from a cloned Drosophila melanogaster (fruit fly) gene, Rdl (resistant to dieldrin), was used to investigate localization of a GABA receptor subunit in adult male D. melanogaster. Many regions in the brain and thoracic ganglia were stained with this antibody. For example, staining was detected in the medulla, lobula and lobular plate optic neurpiles. Also stained were the antennal lobe glomeruli, the ellipsoid body of the central complex and the mushroom bodies. These results suggest possible roles for an RDL-like GABA receptor subunit in the processing of olfactory, visual and mechanosensory information in the nervous system of D. melanogaster.

Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly. The staining of a discrete group of neurosecretory cells in the pars intercerebralis of the brain indicates a possible new role for this mAChR in the regulation of neurosecretion. Very little staining is detected in the thoracic nervous system.

Objective: The authors determined whether there was an advantage to laparoscopic appendectomy when compared with open appendectomy. SUMMARY/BACKGROUND DATA: The advantages of laparoscopic appendectomy versus open appendectomy were questioned because the recovery from open appendectomy is brief., Methods: From January 15, 1992 through January 15, 1993, 75 patients older than 9 years were entered into a study randomizing the choice of operation to either the open or the laparoscopic technique. Statistical comparisons were performed using the Wilcoxon test., Results: Thirty-seven patients were assigned to the open appendectomy group and 38 patients were assigned to the laparoscopic appendectomy group. Two patients were converted intraoperatively from laparoscopic appendectomies to open procedures. Thirty-one patients (81%) in the open group had acute appendicitis, as did 32 patients (84%) in the laparoscopic group. Mean duration of surgery was 65 minutes for open appendectomy and 87 minutes for laparoscopic appendectomy (p < 0.001). There were no statistically significant differences in length of hospitalization, interval until resumption of a regular diet, or morbidity. Duration of both parenteral and oral analgesic use favored laparoscopic appendectomy (2.0 days versus 1.2 days, and 8.0 days versus 5.4 days, p < 0.05). All patients were instructed to return to full activities by 2 weeks postoperatively. This occurred at an average of 25 days for the open appendectomy group versus 14 days for the laparoscopic appendectomy group (p < 0.001)., Conclusions: Patients who underwent laparoscopic appendectomies have a shorter duration of analgesic use and return to full activities sooner postoperatively when compared with patients who underwent open appendectomies. The authors consider laparoscopic appendectomy to be the procedure of choice in patients with acute appendicitis.

The pharmacological properties of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) were investigated using two independent transient expression systems. The binding characteristics of the expressed receptor were determined using transfected COS-7 cells, whereas the mAChR functional properties were analyzed using nuclearly injected Xenopus oocytes. Competition displacement studies with transfected COS-7 cell membranes showed that N-[3H]methylscopolamine binding was displaced most effectively by atropine, followed by 4-diphenylacetoxy-N-methylpiperidine methiodide, pirenzepine, and AFDX-116. This same order of effectiveness (4-diphenylacetoxy-N-methylpiperidine methiodide > pirenzepine > AFDX-116) was observed in oocytes expressing Dm1 when carbamylcholine-induced currents were inhibited by the same antagonists. Thus, the expressed Drosophila mAChR (Dm1) exhibits a pharmacology that broadly resembles that of the vertebrate M1 and M3 mAChR subtypes. To determine the anatomical localization of the Drosophila mAChR, polyclonal antiserum was raised against a peptide corresponding to the predicted carboxyl-terminal domain of the receptor. Immunocytochemistry on fly sections demonstrated that the mAChR gene product was found in the nervous system and was not seen in skeletal muscle. The most intense staining was localized to the glomeruli of the antennal lobes, an area of the insect brain where first-order synaptic processing of olfactory information occurs.

Receptors for 4-aminobutyric acid (GABA) have been identified in both central and peripheral nervous systems of several invertebrate phyla. To date, much of the information derived from physiological and biochemical studies on insect GABA receptors relates to GABA-gated chloride channels that show some similarities with vertebrate GABAA receptors. Like their vertebrate central nervous system (CNS) counterparts, agonist activation of such insect GABA receptors leads to a rapid, picrotoxin-sensitive increase in chloride ion conductance across the cell membrane. In insects, responses to GABA can be modulated by certain benzodiazepines and barbiturates. However, recent studies have detected a number of striking pharmacological differences between GABA-gated chloride channels of insects and vertebrates. Receptor binding, electrophysiological and 36Cl- flux assays have indicated that many insect receptors of this type are insensitive to the vertebrate GABAA antagonists bicuculline and pitrazepin. Benzodiazepine binding sites coupled to insect GABA receptors display a pharmacological profile distinct from that of corresponding sites in vertebrate CNS. Receptor binding studies have also demonstrated differences between convulsant binding sites of insect and vertebrate receptors. Insect GABA receptor molecules are important target sites for several chemically-distinct classes of insecticidally-active molecules. By characterizing these pharmacological properties in detail, it may prove possible to exploit differences between vertebrate and insect GABA receptors in the rational design of novel, more selective pest control agents. The recent application of the powerful techniques of molecular biology has revealed a diversity of vertebrate GABAA receptor subunits and their respective isoforms that can assemble in vivo to form a multiplicity of receptor subtypes. Molecular cloning of insect GABA receptor subunits will not only enhance our understanding of invertebrate neurotransmitter receptor diversity but will also permit the precise identification of the sites of action of pest control agents.

The role of septohippocampal circuits in the generation of the P300 response in cats (n = 12) was explored in a series of depth recording, tract-tracing and lesion experiments. Systematic mapping of the hippocampus in 1-mm increments from rostral to caudal extent revealed large positive potentials, greater in amplitude to rare than to frequent stimuli, within the 200-500 ms range. Each map revealed maximal amplitude responses at diverse, widely distributed hippocampus loci. Furthermore, these electrical responses displayed polarity inversion within the hippocampus that was generally localized to the pyramidal cell layer; polarity inversion was also observed in the adjacent entorhinal cortex and amygdala. Injections of propidium iodide, a tract-tracing agent, into these inversion sites resulted in retrograde labeling of small clusters of choline acetyltransferase (ChAT)-positive neurons in the medial septal nucleus and vertical limb of the diagonal band. Aspiration lesions that bilaterally destroyed large amounts of caudal hippocampus from stereotaxic levels A4 to A1 resulted in a decreased number of cells expressing ChAT in the rostral basal nuclear complex. In only 2 cats was the preoperative presence of a significant vertex P300 absent postoperatively. In the majority of cases (5 of 8 animals), hippocampal aspiration produced an enhancement of the preoperative P300 potential. We conclude that cholinergic mechanisms are importantly, albeit not exclusively, involved in the mediation of P300 potentials in cats. Neurons mediating P300 responses appear to be organized in diverse clusters of septal and diagonal band cells. These septal cells may facilitate, and in turn be facilitated or inhibited as a function of hippocampal, or other, allocortical feedback loops.

We have investigated the fate of the large amounts of calcium ingested by Rhodnius prolixus in its meals of blood. 45Ca2+ injected into the haemolymph or fed to fifth-stage Rhodnius reared on rabbits is accumulated at high concentrations in the cells of the upper Malpighian tubules; very little is excreted from the body This 45Ca2+ accumulation goes on continuously for at least 12 days and the rate of uptake is increased several-fold within 3-4 days of a meal. The extent of calcium accumulation in tubule cells is correlated with the presence of intracellular membrane-bound concretion bodies, which are therefore likely sites of calcium deposition. X-ray diffraction showed that the calcium deposits are non-crystalline. Tubules from rabbit-fed fifth-stage Rhodnius contain 410 mmol l-1 calcium; in those from chicken-fed insects the calcium concentration is over 1 mol l-1; and in those fed in vitro on heparinised low-K+ sheep blood the calcium concentration is only 21 mmol l-1. The concentration of calcium in the haemolymph in all these insects was 8 mmol l-1 and its activity determined by an ion-selective electrode was 2.5 mmol l-1. 45Ca2+ deposited in the tubules is readily exchangeable, but the efflux preferentially passes to the haemolymph side of the tubule epithelium. The ability to sequester calcium in the Malpighian tubules may prevent calcium from interfering with reabsorptive processes in the rectum.

Immunoreactivity for choline acetyltransferase (ChAT) was analyzed in unoperated cats and in cats in which stereotaxic lesions were made in the pedunculopontine and laterodorsal tegmental nuclei. The fine reaction product revealed moderate to dense ChAT-immunoreactive fiber plexuses throughout the telencephalon, diencephalon, and midbrain. A pontomesencephalic origin of cholinergic innervation to virtually every nucleus of the diencephalon, as well as to various midbrain and basal telencephalic sites was indicated in the cats with lesions, in which the optical density of ChAT-immunoreactivity was significantly decreased as compared to controls. Pontomesencephalic lesions produced no changes, however, in the density of ChAT staining in the cerebral cortex, basolateral amygdala, or caudate nucleus. In addition to ChAT-positive terminal fiber arborizations which were widely distributed, cholinergic fibers-of-passage were traced in the unoperated and operated feline brains. The general course of ChAT fibers cut in cross-section was followed in successive transverse levels, and although pathways originating from the pedunculopontine nucleus demonstrated orientations in every direction, many demonstrated a rostral course. A particularly dense aggregate of ascending ChAT-positive fibers was localized in the dorsolateral sector of the pedunculopontine area which could be followed at more rostral levels into the central tegmental fields and the compact part of the substantia nigra. From the central tegmental fields, numerous ChAT-immunopositive fibers cut in cross-section continued to course rostrally in the intralaminar, reticular and lateroposterior nuclei of the thalamus, and a distinct bundle of ChAT fibers coursing dorsolaterally was observed medial to the optic tract ascending to the lateral geniculate. ChAT fibers with dorsolateral orientations were additionally observed in the zona incerta, ventral anterior thalamus, and ansa lenticularis on route to the reticular thalamus, the globus pallidus, and the substantia innominata. Pathways consisting of fibers traced from ChAT-containing cells in the laterodorsal tegmental nucleus could be traced to medial structures such as the periaqueductal gray, ventral tegmental area and dorsal raphe. Medially placed ChAT fibers were additionally followed through the ventral tegmental area, the midline thalamus, and the hypothalamus, up to the medial and lateral septal nuclei. The trajectories of the ascending cholinergic pathways from the pontomesencephalon are discussed in relation to locally generated electrophysiological responses in the cat.

Wave A in the cat appears to be analogous to P1 in the human. Both are positive middle-latency auditory-evoked potentials, present at slow click rates during wakefulness and REM sleep but absent during slow-wave sleep. Wave A has been recorded in the parabrachial and medial tegmental areas of the midbrain and in thalamic target projections of the reticular activating system. Two nuclei in this system, the pedunculopontine tegmental (PPT) and laterodorsal tegmental (LDT) nuclei, contain cholinergic cells; the cholinergic antagonist scopolamine eliminates Wave A. To test whether PPT and LDT were important in Wave A generation, we attempted to lesion these nuclei bilaterally in 11 cats. Wave A was markedly diminished or absent in all but 2 cats, in which the lesions did not include PPT. Loss of choline acetyltransferase-positive cells in PPT, but not LDT, was correlated with effects on Wave A, i.e. greatest cell loss occurred in cats in which Wave A disappeared, and least cell loss in cats with no change in Wave A. We conclude that the PPT nucleus, and particularly its cholinergic cell component, is essential for Wave A generation and suggests that a similar substrate may be significant for generation of the human P1.

The cat-P300 is a positive endogenous potential, larger to a stimulus when rare than when frequent, with a latency of 200-500 msec. The role of polysensory association cortex, postulated to be important in human P300 generation, was assessed in the cat. EEG was recorded in 13 awake cats from a skull screw at the vertex. Stimuli included frequent (P = 0.80) 1 kHz and rare (P = 0.10) 2 kHz tone pulses with probabilities counterbalanced across 260-trial blocks. After 12 preoperative sessions, bilateral ablations were made of pericruciate cortex (4 cats), anterior lateral and medial suprasylvian gyri (4 cats) and all 3 areas (5 cats). Postoperatively, all 13 cats showed a P300 across 12 recording sessions. Thus polysensory association cortex is not essential for generation of the cat-P300.

This paper provides a detailed description of the dissociation and maintenance in culture of cells derived from embryonic tissue and from the central nervous system of adult housefly, Musca domestica. Dissociation of embryonic tissue produced several cell types of which muscle and neurone-like cells predominated. These cells were morphologically distinct once development had started in vitro with muscle cells often producing rhythmic contractions. Neurones had small cell bodies (less than 10 mum) and showed extensive outgrowth of neurites. Neuronal cell bodies readily sealed onto patch pipettes and with pipette solutions containing nicotinic agonists, single channel currents could be recorded. With cell-attached patches, these currents were inward at the cell resting potential and reversed with depolarization. Channels with two different conductances could be seen in the majority of patches.

The hepatopancreas of the lobster has recently been found to be a rich source of material from which to isolate arthopod gap junctions biochemically (Finbow et al., 1983a; 1984). It has therefore been studied here to assess the features of these intercellular junctions and any others that may be present, in vivo. The tissue consists of columnar epithelial cells which possess apical microvilli and basal infoldings. In thin sections the lateral borders of these cells are characterized by desmosomes and smooth septate junctions as well as by gap junctions. The desmosomes exhibit no apparent freeze fracture profile but the septate junctions display parallel rows of ridges or aligned intramembranous particles (IMPs) with complementary grooves on the other membrane half; these IMPs shift in their preferential fracturing plane depending on whether the tissue has first been fixed, always remaining on the EF if unfixed. The IMPs or connexons, of which the gap junctions are composed, fracture onto the E face, leaving complementary pits on the P face, regardless of whether the tissue is fixed or not. At the base of the pancreatic cells, the lateral borders are thrown into interdigitating folds which display endocytotic profiles and possible internalization of junction-bearing membranes. This phenomenon, which is readily visualized both after tracer incubation and in replicas, may represent junctional degradation relating to membrane turnover.

The occurrence of an unusual double plasma membrane structure is reported; it has been studied in conventional thin sections, after lanthanum-impregnation and with freeze-fracturing. This modification of the plasmalemma is found where the luminal cell membrane (I membrane) of gut microvilli in the haematophagous insect, Rhodnius prolixus, is surrounded by a second, outer membrane (O membrane), the 2 separated from one another by a highly regular I-O space of about 10 nm. Lanthanum impregnation reveals the presence of columns inclined at an angle, within this I-O space; as in the continuous junctions which link the lateral borders of these cells, these columns may maintain the very precise I-O distance. From the outer microvillar membranes radiate short spoke-like fibrils or sheets which encounter another more extensive system of myelin-like sheets. Freeze-fracturing reveals that the spoke-like sheets and the other ones which lie like a tube, around and parallel to the microvilli, contain linear ridges composed of particles, lying at random within layers of the myelin-like material which also extends into the lumen of the gut. The microvillar membanes, both O and I, fracture into faces containing rows of either PF particles or EF pits arranged as spiral ridges or grooves around the sides and across the tip of each microbillus. These could be the insertion sites of one or both of the I-O columns and spoke-like sheets while the sheets could represent a variant of peritrophic membrane. The double membrane may be a cellular device to increase the strength of the microvillar layer in these blood-sucking animals, since the cell layer must withstand great pressure owing to a sudden massive extension of the gut during a blood meal.

Endogenous responses were recorded from 9 awake cats with loud and soft clicks randomly presented as rare (P = 0.15) or frequent (P = 0.80) stimuli; a reinforced tone CS (P = 0.05) resulted in a conditioned eye blink response and focused the cat's attention on the auditory stimuli. Subsequent to 12 preoperative recording sessions the medial septal area was lesioned in 7 cats and similar but more rostral lesions were placed in 2 cats. Thereafter, 12 postoperative recording sessions were carried out, the animals were terminated, and the brains processed for AChE histochemistry and histology of the lesioned areas. Destruction of the medial septum and vertical limb of the diagonal band of Broca resulted in a transient postoperative 'cat P300' followed by reduction and disappearance of the response. The hippocampus of these animals was characterized by marked AChE depletion. In contrast, the animals with lesions rostral to the medial septal area showed no postoperative change in the P300 response and no depletion of hippocampal AChE. These data indicate an important role for the medial septal area as a modulator of 'cat P300' generation possibly through the cholinergic component of the septohippocampal projection system.

Smooth septate junctions in the midgut of Musca domestica and in Malpighian tubules of both Musca and Rhodnius prolixus are described. Details of the structures revealed after standard fixation, fixation in the presence of the stain, lanthanum hydroxide, and after freeze-fracture are discussed in the light of models previously put forward to explain the interrelations of the images obtained by these different methods. The organization of the junction between cells of the midgut varies in the apical-to-basal axis. At the apical border the septa (or ridges in freeze-fracture replicas) are packed tightly and follow an undulating but strictly parallel course. This packing loosens towards the middle of the junction until, at its basal extremity, the septa (ridges in replicas) are widely separated and follow independent meandering courses. That these features are found both in lanthanum-infiltrated specimens and freeze-fracture replicas allows a correlation to be made between the septa and the freeze-fracture ridges. The functional significance of these smooth septate junctions is discussed.

The purpose of this study is to look for structural correlates of the demonstrated nicotine-insensitivity of larval Manduca sexta CNS, an insensitivity which is only slightly perturbed by desheathing (a technique used to disrupt perineurial diffusion barriers). The general organization of the hornworm ganglion is found to conform to the conventional insect pattern, but the following points are noted and discussed in terms of their potential relationship to nicotine-insensitivity: the damage caused to perineurial cells by desheathing is extremely localized, with cells immediately adjacent to the torn region showing good ultrastructural integrity; ionic lanthanum does not gain access to the subperineurial extracellular space following desheathing; lanthanum penetrates the ganglion in the cytoplasm of tracheal cells damaged peripherally during desheathing, but is excluded from the extracellular space surrounding such tracheal cells; smooth endoplasmic reticulum is much in evidence in perineurial cells and tracheal cells, sites where it might be implicated in nicotine detoxification; individual basal perineurial cells appear to cover extensive regions of the ganglion, thereby limiting intercellular diffusion.

The effects of the 27 X 10(3) Mr insecticidal delta-endotoxin from Bacillus thuringiensis var. israelensis have been studied using, as a model system, isolated insect Malpighian tubules. At all concentrations of the toxin higher than 1 microgram ml-1 (4 X 10(-8) moll-1) applied to the outer surface of the tubules, fluid secretion failed within about 30 min. Except at very high concentrations, where failure always takes at least 30 s, there was an inverse relationship between the concentration of toxin and the time of failure of toxin-treated tubules. During exposure to toxin, the tubules were initially unaffected for a relatively long period and then rapid failure occurred. If the tubules were removed into toxin-free saline just before failure would have occurred, fluid secretion remained normal for at least 2 h, but on return to the origin toxin-containing saline failure was almost immediate. The toxin was found not to bind to the basement membrane. Ultrastructural changes became evident as tubule failure occurred. These initially involved modifications to the basal side of the cells, but later also to the luminal microvilli. Intercellular junctions became disassociated and cytoplasmic vacuolization occurred. The population of intramembranous particles in the basal membranes became reduced with time. Our findings suggest the following hypothesis for the initial stages in the interaction of the toxin with the tubules. Toxin molecules attach to the accessible cell membranes progressively and irreversibly. They do not readily associate by diffusing laterally in the membrane, so that toxic effects develop only when sufficiently large numbers of them attach close together. The molecules may then associate in some way as a complex, perhaps forming a pore in the membrane. Relatively few such pores lead rapidly to cell failure and death.

Electrical measurements of membrane potential and resistance using intracellular microelectrodes showed that the fluid-secreting parts of the Malpighian tubules of Rhodnius have superficial regions different in electrical properties from the main body of the cell. The membrane potential in these superficial regions was smaller by 30-40 mV, but showed a standard depolarization on changing the potassium concentration of the bathing medium. The response to changes in the external chloride concentration also differed in the two regions, a finding that was reinforced by different responses to the drug, furosemide. Electron microscopy of the basal regions of the cells revealed many long cellular projections that run parallel to the cell surface and interdigitate with similar projections from neighbouring cells. The degree of interdigitation was examined by marking individual cells with alcian blue or by horseradish peroxidase injection. A survey of the published micrographs of insect Malpighian tubules shows that most have similar projections on their basal surfaces and not the simple basal infoldings previously supposed.

Intracellular recordings demonstrated a transfer of impulses between the paired giant axons of Sabella, apparently along narrow axonal processes contained within the paired commissures which link the nerve cords in each segment of the body. This transfer appears not to be achieved by chemical transmission, as has been previously supposed. This is indicated by the spread of depolarizing and hyperpolarizing voltage changes between the giant axons, the lack of effects of changes in the concentrations of external divalent cations on impulse transmission and by the effects of hyperpolarization in reducing the amplitude of the depolarizing potential which precedes the action potentials in the follower axon. The ten-to-one attenuation of electronic potentials between the giant axons argues against the possibility of an exclusively passive spread of potential along the axonal processes which link the axons. Observation of impulse traffic within the nerve cord commissures indicates, on the other hand, that transmission is achieved by conduction of action potentials along the axonal processes which link the giant axons. At least four pairs of intact commissures are necessary for inter-axonal transmission, the overall density of current injected at multiple sites on the follower axon being, it is presumed, sufficient to overcome the reduction in safety factor imposed by the geometry of the system in the region where axonal processes join the giant axons. The segmental transmission between the giant axons ensures effective synchronization of impulse traffic initiated in any region of the body and, thus, co-ordination of muscular contraction, during rapid withdrawal responses of the worm.

In embryological terms the anal papillae are the product of eversion of the hindgut tissues. The rectum and the anal papillae have the same origin and have a marked structural similarity. The insect hindgut is very labile being able to produce salt transporting or 'chloride cells' from any of the tissues of which it is composed. The hindgut consists of four distinct regions: the ileum and part of the anal canal have a mechanical function, the rectum and the posterior anal canal contain transporting cells. Two new types, 'interstitial' and 'tertiary' are reported in the rectum. The structure of the anal papillae changes with increased salinity. Changes in the plasma membranes alter the surface area for transport. Changes in the number of mitochondria are not accompanied by changes in oxygen consumption. If mitochondria are the site of oxidative metabolism then their number does not control the level of oxygen consumption. In Aedes aegypti the papillary epithelium appears to be a syncytium. Across the lumen of the papillae there are cellular sheets supporting the tracheoles. At the base of the papillae there is a cellular transition zone; circular muscles in this region may be used to occlude the papillae. The control of salt transport may be hormonal.

India ink and ionic lanthanum injections have revealed that the central nervous system (CNS) of the scorpion possesses a highly vascularized cephalothoracic ganglionic mass. It, together with other abdominal ganglia which form a ventral nerve cord, are all ensheathed by an outer layer of modified glial, or perineurial, cells. These cells resemble those which line the blood channels permeating the CNS, in exhibiting both inverted gap and tight junctions. Although the latter show close or fused membrane appositions, lanthanum appears to penetrate past a number, but not all, of them. Freeze-fracturing reveals that these junctions are composed of E-face particles aligned into a network of rows, or ridges, which are frequently discontinuous, especially near the periphery of the perineurium. This produces a somewhat 'leaky' system but occlusion to tracers occurs ultimately, for in the CNS none can be found beyond the perineurium. The existence of this perineurial blood-brain barrier is also demonstrable electrophysiologically where cations such as Mg2+ are unable to penetrate beyond the perineurial layer although they can, it seems, leak in via the blood vascular system. Relative differences in tightness between the perineurium and the cells lining the blood channels may be attributed to differences in the relative number of discontinuous ridges. This is borne out by the observation that the peripheral nervous system has a highly attenuated perineurium with many fewer junctions, and some of these nerves tend to be leaky with respect to tracer penetration. In fixed material the junctional ridges may fracture on to the E-face or partly on both the EF and PF, while in unfixed tissue they are usually found on the PF. In both cases they exhibit complementary grooves that are coincident with the ridges across membrane transitions; in such cases the cell membranes are fused with concomitant obliteration of the intercellular space. These tight junctions, often closely associated with EF gap junctional particle aggregates which may be very loosely clustered, appear to form the basis of the observed blood-brain barrier in the scorpion CNS.

Tracer and freeze-fracture techniques are used to evaluate the capacity of the central and peripheral nervous system of the horseshoe crab, Limulus polyphemus to admit or exclude molecular or ionic constituents of the blood intercellularly. Both the peripheral and central nervous systems are contained within blood sinuses so there is intimate contact between the haemolymph and the neural lamella. No discrete perineurium exists so any protection afforded to the nerve cells must be provided by the ensheathing glial cells and any junctions between them. Using ionic lanthanum as a pre-fixation incubation medium the system is seen to be completely "open', with the tracer gaining access to all regions of the nervous tissue. Cellular association in the peripheral nervous system, as revealed by thin-section and freeze-fracture, consist only of small scattered gap junctions between glial cells which afford no restriction to tracer entry. Gap junctions are again present between glial cells in the C.N.S. but here they are far more numerous, sometimes forming extensive sheets of almost continuous gap junctional plaques. Between certain glial cells there also exists a junctional system of linear PF ridges and complementary EF grooves; these may associate with or surround, often discontinuous arrays, the gap junctional plaques. Given their characteristics and the freedom of tracer entry, they seem unlikely to represent either typical occluding tight junctions or septate junctions.

This paper describes the structural characteristics and permeability properties of the smooth septate junctions between the upper Malpighian tubule cells of a blood-sucking bug, Rhodnius prolixus. The permeability of the paracellular route was tested only for solutes that could be demonstrated not to cross the epithelium via the cellular route. The intercellular clefts were readily permeated by sucrose, inulin and polyethylene glycol (PEG), showing a higher permeability to molecules of smaller radius (PEG versus sucrose). Negatively charged molecules permeated the clefts more readily than positively charged ones. The effects of pH, urea and luminal flow rate on permeability were studied. The results are discussed in relation to the physiological tightness of the Malpighian tubules to certain solutes and to its function as an excretory epithelium.