Immunohistochemical determination of the site of antidepressant-like effects of glucagon-like peptide-2 in ACTH-treated mice.

The intracerebroventicular administration (i.c.v.) of glucagon-like peptide-2 (GLP-2) had antidepressant-like effects on saline-treated mice in the forced-swim test. The GLP-2 treatment (3 µg, i.c.v.) for 6 days, but not that of imipramine had antidepressant-like effects on adrenocorticotropic hormone (ACTH)-treated mice. The immunohistochemical detection of the c-fos protein (Fos) revealed that the administration of GLP-2 induced Fos-immunoreactivity (Fos-IR) in the dorsomedial hypothalamic nucleus in saline-treated and ACTH-treated mice, and also in the hippocampal dentate gyrus in ACTH-treated mice, but not in saline-treated mice. In contrast, Fos-IR in the paraventricular nucleus of the hypothalamus decreased after the administration of GLP-2 to ACTH-treated mice. In ACTH-treated mice, the chronic administration of GLP-2 affected hippocampal neurogenesis, in addition to Fos-IR in hypothalamic GABAergic neurons and corticotrophin-releasing factor-containing neurons. These results suggest that GLP-2 acts on specific brain regions to regulate stress conditions, and induces antidepressant-like effects under imipramine-resistant conditions, which may be associated with the modulation of the hypothalamic-pituitary-adrenal-axis.

Diabetes onset influences hippocampal synaptic plasticity in streptozotocin-treated rats.

Children with type 1 diabetes mellitus (DM) are at risk of developing cognitive difficulties. Although a diabetes onset of patient influences cognitive difficulties, synaptic properties related to the age of diabetes onset remain unknown. Here we showed that synaptic plasticity including long-term potentiation (LTP) or long-term depression (LTD), and excitatory synaptic transmission at Schaffer collateral-CA1 (SC-CA1) synapses in hippocampal slices were affected by age of onset in rats with streptozotocin-induced diabetes (STZ-rats), compared with age-matched control rats. LTP was impaired and the ratio of AMPA receptor-mediated EPSCs relative to N-methyl-d-aspartate (NMDA) receptor-mediated EPSCs (the AMPA/NMDA ratio) decreased in young adult-onset STZ-rats, whereas LTD was impaired and both AMPA receptor-mediated and NMDA receptor-mediated EPSCs increased in juvenile-onset STZ-rats. Furthermore, impaired LTD of juvenile-onset STZ-rats was restored with an NMDA receptor antagonist. These results suggest that the pathophysiology of diabetes-induced cognitive difficulties varies with the age of diabetes onset.

Immunohistochemical determination of the site of hypotensive effects of glucagon-like peptide-2 in the rat brain.

Proglucagon-derived glucagon-like peptide-2 (GLP-2) is released from enteroendocrine cells and neurons. GLP-2 regulates energy absorption and epithelial integrity in the gastrointestinal tract, but its effect on blood-pressure regulation remains unknown. In the present study, we found that GLP-2 administered both peripherally and centrally dose-dependently reduced mean arterial blood pressure (MAP) in male Wistar rats anesthetized with urethane and α-chloralose. Immunohistochemical detection of the c-fos protein (Fos) revealed that the peripherally and centrally administered GLP-2 induced Fos-immunoreactivity (Fos-IR) in the nucleus of the solitary tract (NTS) and the caudal ventrolateral medulla (CVLM). In contrast, Fos-IR in brainstem catecholamine neurons decreased after the administration of GLP-2. These results suggest that GLP-2 acts on specific brain nuclei to inhibit sympathetic nerve activity and this leads to hypotension.

A new technique for implanting a fine-wire microelectrode for chronic recording of unit activity from freely-moving mice.

We report here a newly developed chronic implantation technique using an epoxy-coated fine-stainless steel wire (33 microm in diameter) to record single unit activity from the brain of freely-moving mice with as little tissue injury as possible. Since the fine-wire electrode is not capable of staying straight by itself or of penetrating into the brain, a pair of permanent neodymium magnets placed on a micromanipulator as well as below the animal's head was used for stereotaxic implantation to keep the fine-wire straight and strong by the magnetic fields. With those implanted electrodes recording of single units from the hippocampal CA1 of freely-moving mice was performed during sleep and wakefulness.

Glucagon-like peptide-1 modulates neuronal activity in the rat's hippocampus.

In order to assess effects of glucagon-like peptide-1 (GLP-1) in the brain excluding the hypothalamus, the effects of GLP-1 (7-36) amide, a naturally produced active fragment, on the electroencephalogram and hippocampal single unit activities of anesthetized male Wistar rats were examined. I.c.v. injection of GLP-1 (7-36) amide decreased the hippocampal theta wave duration. Juxtacellular administration of GLP-1 (7-36) amide first increased and then decreased single unit activities recorded in the hippocampal CA1, which effects were antagonized by exendin (9-39) amide, a GLP-1 receptor antagonist, or 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA type glutamate receptor antagonist. These results indicate that GLP-1 receptors exist in the hippocampus and are involved in modulating hippocampal activity through an increase in the release of excitatory amino acid transmitters.

Dorsal spinocerebellar tract neuronal activity in the intact chronic cat.

The ability to electrophysiologically identify the axonal projections of lumbar neurons recorded in chronic unanesthetized intact awake animals is a formidable but essential requirement toward understanding ascending sensory transmission under naturally occurring conditions. Chronic immobilization procedures previously introduced by Morales et al. (1981) for intracellular studies of motoneurons are modified and then integrated with procedures for antidromic cellular identification and extracellular recording of upper (or lower) dorsal lumbar spinocerebellar tract (DSCT) neuronal activity, in conjunction with behavioral state recording and drug microiontophoresis. These implant procedures provide up to 6 months of stable recording conditions and, when combined with other techniques, allow individual DSCT neurons to be monitored over multiple cycles of sleep and wakefulness, following the induction into and recovery from barbiturate anesthesia and/or during the juxtacellular microiontophoretic ejection of inhibitory or excitatory amino acid neurotransmitters. The combination of such techniques allows a comprehensive examination of synaptic transmission through the DSCT and other lumbar sensory pathways in the intact normally respiring cat and its modulation during the general anesthetic state. These techniques permit investigations of the supraspinal controls impinging on lumbar sensory tract neurons during wakefulness and other behavioral states such as active sleep.

Synaptic transmission through cat lumbar ascending sensory pathways is suppressed during active sleep.

1. Few data are available that describe the evoked activity of spinal cord sensory tract neurons as a function of behavioral state. Accordingly, experiments were performed in which ascending volleys were recorded extracellularly within the spinoreticular (SRT), spinothalamic (STT), and spinomesencephalic (SMT) tracts located in the ventrolateral reticular formation in response to low-intensity electrical stimulation of the contralateral sciatic nerve in the chronic unanesthetized, behaving cat during naturally occurring episodes of wakefulness, quite sleep, and active sleep. 2. During episodes of wakefulness and quite sleep sciatic nerve stimulation produced a low-amplitude and long-duration orthodromic field potential that did not differ in amplitude or waveform. However, during the corresponding episode of active sleep, the sciatic nerve-induced orthodromic field potential was markedly suppressed or abolished. 3. The effects of sustained microiontophoretic applications of inhibitory amino acid agonists, glycine, or gamma-aminobutyric acid during wakefulness or quite sleep markedly suppressed the antidromic field potential recorded from nearby VII motoneurons but did not alter the sciatic nerve-evoked orthodromic field potential, indicating that the sciatic response was recorded from ascending axons of passage emanating from lumbar spinal neurons. We suggest that lumbar neurons comprising the SRT, STT, and SMT tracts are subjected to a descending suppressor drive that is activated specifically during the behavioral state of active sleep.

NMDA receptors mediate neuronal burst firing in rat somatosensory cortex in vivo.

Two patterns of neuronal firing, bursting and regular spiking, are observed in the somatosensory cortex of anesthetized rats. The effects of excitatory amino acid receptor agonists and antagonists on these discharge types have been examined in vivo, in order to assess their involvement in the generation of such firing patterns. The analysis of interspike interval histograms indicates that N-methyl-D-aspartate (NMDA) receptors are involved in the generation of bursting responses while non-NMDA receptors mediate the regular spiking pattern of firing in individual neurons. It is also suggested that a basal level of ongoing neuronal excitation via the activation of non-NMDA receptors is required for bursting to be evoked through NMDA receptors.

GABAA receptor-induced inhibition of neuronal burst firing is weak in rat somatosensory cortex.

Extracellular and intracellular recordings have been made from the somatosensory cortex of anesthetized rats. From 264 neurons having receptive fields including the contralateral whiskers, we have determined that two distinct patterns of firing are observed, and call these two patterns regular spiking and bursting. Iontophoretically administered gamma-aminobutyric acid (GABA), muscimol, or flurazepam showed significantly weaker inhibition of the bursting pattern of firing than of the regular spiking pattern. By contrast, baclofen inhibited both types of firing to the same extent. These results suggest that intracortical GABA receptor subtypes are involved in different ways in modulating the expression modes of discharge.

Amino acids as transmitters of synaptic excitation in neocortical sensory processes.

Few synaptic transmitters are known to exist that are not represented in some region or another, or at some layer or other, in the cerebral cortex of mammalian brain. The more difficult job than mere identification of which substances are present, is that of the assignment of particular functional role(s) of such substances, and as well, of determining upon exactly which element(s) of the known synaptic circuitry of neocortex, such transmitters operate. Current wisdom subscribes to the view that the excitatory amino acids, most likely L-glutamate, and L-aspartate but perhaps also L-cysteate, L-homocysteate, L-cysteine sulfinate or even (although much less likely) the endogenous dipeptide substance, N-acetyl-L-aspartyl-L-glutamate, are the major excitatory synaptic transmitters of intracortical (associational) fibres, of corticofugal projections, and, as this article will attest, of thalamocortical inputs, as well. What particular limits, or restrictions, are imposed upon these generalizations, such as whether the data pertain only to primary sensory areas or follow some other yet to be determined rule, remains to be discovered in future experiments. This paper first presents an overview of the advances in understanding that have come about during the past few decades concerning the synaptic roles of amino acid transmitters. Next, an experimental section presents new evidence based on release studies and the microiontophoretic approach, which supports the view that the amino acids, glutamate and aspartate, interact with specific, pharmacologically identified subtypes of receptors in neocortex as transmitters of synaptic excitation released from thalamic afferent terminals.

Benzodiazepines and synaptic processing in the spatial domain within the cat's primary somatosensory cortex.

In the primary somatosensory cortex of cats, the size of the receptive fields (RFs) of cutaneously responsive neurones is under the control of gamma-aminobutyric acid (GABA) mediated inhibition when the cells are situated in rapidly adapting (RA) background regions. Cells located in slowly adapting (SA) or low-velocity rapidly adapting (LVRA) background regions do not appear to be affected by GABA significantly in the spatial domain, although other response properties such as threshold and firing pattern are under the influence of bicuculline methiodide (BMI) sensitive processes. The GABA receptor is one component of the oligomeric complex that includes the benzodiazepine (Bzd) binding site, the barbiturate recognition site, and the Cl- ionophore. Owing to current debates about the possible existence of endogenous ligands of Bzd receptors, we have examined whether Bzd agonists, in addition to GABA and BMI, have RF-modulating actions on RA S1 neurones and have assessed the effectiveness of the Bzd antagonist, Ro 15-1788, in this experimental paradigm. Ro 15-1788 is an imidazobenzodiazepine that acts as a specific competitive antagonist of Bzds by exerting high-affinity interactions with that Bzd receptor through which anticonvulsant effects of flurazepam (flu) and diazepam are expressed. This has been shown previously in neurochemical, behavioral, neurological, and pharmacological studies. Ro 15-1788 has little or no affinity for nonneuronal binding sites in the CNS. Ro 15-1788 binding does not displace GABA from its own binding site but does compete for all major Bzd ligands that act as pharmacological agonists and inverse agonists of the Bzd receptor through which anticonvulsant and convulsant effects are expressed. Bzd agonists elevated the threshold for somatic activation, depressed spontaneous activity, and decreased RF size. One exception in this regard was midazolam, which sometimes decreased somatic thresholds and increased spontaneous discharges. These latter effects were reversed at higher doses of the agonist. BMI returned RFs to control sizes when the drug was administered concurrently with Bzd agonists, or it caused RFs to assume greater than normal sizes, depending on the strength of current ejecting the antagonist. Ro 15-1788 given alone decreased response thresholds, increased spontaneous firing, and sometimes enlarged RFs. This antagonist also reversed the RF size-decreasing action of flu, diazepam, and midazolam. Quantitative analyses of air-puffer responses evoked from low-threshold, S1 cells revealed that Bzds do not selectively attenuate spatial summation, but that they act preferentially in the surround, or in the peripheral, regions of cutaneous excitatory RFs.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptive-field size of S1 cortical neurones is altered by methaqualone via a GABA mechanism.

Methaqualone (Mtq; quaaludes or 'ludes) is a controlled substance, having a molecular structure related to the imidiazobenzodiazepine series of drugs, that has gained some notoriety recently due to its history of widespread abuse on the street. Users report experiencing peripheral paresthesia and transient numbness on body parts receiving dense cutaneous innervation (lips, fingertips, etc.). Since the receptive-field (RF)-sizes of many primary somatosensory (S1) cortical neurones are controlled by local, gamma-aminobutyric acid (GABA)-mediated inhibitory processes, we tested Mtq to see whether its clinical symptoms might have a basis in an action through central GABA-mediated synaptic processes. This report supports this contention and describes a likely pharmacological mechanism involved as one being related to the Ro 15-1788-sensitive benzodiazepine (Bzd) recognition site(s) of the GABA receptor complex.

Calcium-dependent dynamic changes in the subcellular distribution of calmodulin in frog spinal cells.

Calcium-dependent changes in the subcellular distribution of calmodulin (CaM) were investigated using isolated intraarterially perfused frog spinal cords. Perfusion with quin2-tetraacetoxymethyl ester (Q2-AM), an intracellular Ca2+-chelator, increased soluble CaM, measured by radioimmunoassay (RIA), with a concomitant decrease in particulate CaM. By contrast, in synaptosomes, Q2-AM increased soluble CaM and total CaM but not particulate CaM. These results suggest that intracellular Ca2+ controls the binding of CaM to membranes in spinal cells.

The effects of hemodialysis with different membranes on middle molecules and uremic neuropathy.

Twenty-four hemodialysis patients, 14 with uremic neuropathy and 10 symptom-free, were studied over 12 months. Cuprophan and AN 69 membrane dialyzers were used in their treatment in order to investigate the influence of different membranes on plasma levels of middle molecular weight substances (MMS) and uremic neuropathy. Hemodialysis with the cuprophan membrane caused no significant changes in plasma MMS levels or in the neurological condition of patients. The effect of dialysis with AN 69 membrane depended on initial plasma MMS levels. Initially high plasma MMS levels decreased significantly and significant improvement of neuropathy was achieved. In neuropathic patients with plasma MMS levels similar to those of symptom-free patients, hemodialysis with AN 69 membrane had no effect. These results suggest that hemodialysis with MMS high-permeability membranes may be recommended for neuropathic patients with high plasma MMS levels.

Cellular distribution of protein-disulfide interchange enzyme activity in mouse spleen.

In an attempt to precise the cellular distribution of protein-disulfide interchange enzyme activity within different compartments of the splenic lymphoid tissue, we have analyzed the protein-disulfide interchange (PDI) enzyme activity in adherent and nonadherent cell populations of normal and T-cell depleted CBA mice. In vivo depletion of T-cells, as evaluated by functional and cytotoxic tests, was achieved by two i. v. injections of anti-T monoclonal antibodies (Mab F7D5). Nonadherent cell populations were found to have levels of protein-disulfide interchange enzyme activity significantly higher than that of the adherent cells. Pretreatment with F7D5 monoclonal antibodies enhanced the protein-disulfide interchange activity in nonadherent cell population, thus indicating that the major source of the enzyme activity are nonadherent spleen cells, which do not bear T-cell marker, probably B-cells.

Benzodiazepine receptor involvement in the control of receptive field size and responsiveness in primary somatosensory cortex.

Microiontophoretic ejection of 3 benzodiazepines (BZDs), flurazepam, diazepam and midazolam, and of Ro 15-1788 to primary somatosensory cortical neurons altered the nature of the responses evoked by natural stimulation of the cutaneous surface. BZDs raised the thresholds to tactile stimulation and produced a decrease of the receptive field areas of neurons situated in rapidly adapting submodality regions of S1 cortex; these effects of the BZDs were exerted at different apparent potencies. Ro 15-1788 antagonized the BZD-induced receptive field size decreases and threshold changes. When administered alone, this substance sometimes lowered the threshold for tactile activation and slightly enlarged the receptive field size.

Anisatin, a potent GABA antagonist, isolated from Illicium anisatum.

The neuropharmacological properties of anisatin were tested on the frog spinal cord and the crude synaptic membrane from rat brain. Anisatin (10(-5) M) reduced the amplitude of dorsal root potentials induced by stimulation of the adjacent dorsal root and presynaptic inhibition of the ventral root reflex. Anisatin shifted the dose-response curve for GABA-induced depolarization in the primary afferent terminal to the right and also reduced the maximum response to GABA. [3H]Muscimol binding to the crude synaptic membrane was not inhibited by anisatin. These results indicate that anisatin is a picrotoxin-like, non-competitive GABA-antagonist.

The ontogenetic evolution of acidic phosphoprotein phosphatase activity in the lymphatic tissue and the liver of the rat.

We have shown that an acidic phosphoprotein phosphatase (APP-ase) has a different pattern of postnatal maturation in the spleen, thymus and liver of rats and mice. The APP-ase activity increases during the first eight months of postnatal life in the spleen of rats (when it attains an 8--10 times higher value than at birth) and up to the sixth month of life in the spleen of mice. It increases considerably during the first two weeks of postnatal life in the thymus of rats and mice; in the liver of rats it reaches maximum activity before birth, but continues to increase up to the sixth month of postnatal life in the liver of mice. The results show also that the APP-ase from the spleen, thymus and liver of rats is equally active in dephosphorylating ATP and phenyl phosphate during the whole life span of rats, but not in relation to beta-glycerol phosphate. After analyzing its substrate specificity, its pH dependence in relation to different substrates, its kinetic properties, as well as its behaviour towards ascorbic acid and different inhibitors (sodium tungstate and sodium molybdate, L-tartrate, L-phenylalanine and L-cysteine) we have come to the conclusion that the rat spleen APP-ase is different from "nonspecific" acid and alkaline phosphatases and very similar to the EC 3.1.3.16 acid phosphoprotein phosphatase.