Stereological estimates of the basal forebrain cell population in the rat, including neurons containing choline acetyltransferase, glutamic acid decarboxylase or phosphate-activated glutaminase and colocalizing vesicular glutamate transporters.

The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.

Codistribution of GABA- with acetylcholine-synthesizing neurons in the basal forebrain of the rat.

In recent years, GABAergic neurons have been identified in the basal forebrain where cholinergic cortically projecting neurons are located and known to be important in mechanisms of cortical activation. In the present study in the rat, the relationship of the GABA-synthesizing neurons to the acetylcholine-synthesizing neurons was examined by application of a sequential double staining immunohistochemical procedure involving the peroxidase-antiperoxidase technique for glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT). In these double and adjacent single immunostained series of sections, the GAD+ and ChAT+ cells were mapped, counted and measured with the aid of a computerized image analysis system. Through the entire basal forebrain, there was no evidence for colocalization of GAD and ChAT in the same neurons. Instead, a large population of GAD-immunoreactive neurons is codistributed with ChAT-immunoreactive neurons and outnumbers them by a factor of two: approximately 39,000 GAD+ cells to 18,000 ChAT+ cells. Although the GAD+ and ChAT+ neurons lie intermingled within fascicles of the major longitudinal and transverse forebrain fiber systems in subregions of the basal forebrain, the GAD+ cells are more highly concentrated within different sectors of the pathways and regions than the ChAT+ cells. Although GAD+ neurons resemble ChAT+ neurons in certain regions, both being bi- or multipolar and, on average, medium-sized cells, the GAD+ neurons are, in the majority (51%), small-sized cells (< 15 microns in length) and as a population significantly smaller than the ChAT+ neurons. These results suggest that many GABAergic neurons may represent interneurons in the basal forebrain and potentially exert an inhibitory influence on adjacent cortically projecting cholinergic neurons. Medium- to large GAD+ cells, which resemble similar ChAT+ cells, are also present and represent the majority of the GAD+ cells in the nucleus of the diagonal band of Broca, magnocellular preoptic nucleus, and olfactory tubercle, but represent the minority in the anterior and posterior substantia innominata and globus pallidus. Given their prominent size, such GABAergic cells may also exert an inhibitory influence outside the basal forebrain as projection neurons and potentially in parallel with cholinergic neurons, to certain regions of the cerebral cortex. Accordingly, GABAergic cells may be considered as constituents of the magnocellular basal nucleus and potentially important elements within the ventral extrathalamic relay from the brainstem reticular formation to the cerebral cortex.

Projections of GABAergic and cholinergic basal forebrain and GABAergic preoptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat.

Within the basal forebrain, gamma-aminobutyric acid (GABA)-synthesizing neurons are codistributed with acetylcholine-synthesizing neurons (Gritti et al. [1993] J. Comp. Neurol. 329:438-457), which constitute one of the major forebrain sources of subcortical afferents to the cerebral cortex. In the present study, descending projections of the GABAergic and cholinergic neurons were investigated to the lateral posterior hypothalamus (LHp) through which the medial forebrain bundle passes and where another major forebrain source of subcortical afferents is situated. Retrograde transport of cholera toxin b subunit (CT) from the LHp was combined with immunohistochemical staining for glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT) using a sequential peroxidase-antiperoxidase (PAP) technique. A relatively large number of GAD+ neurons (estimated at approximately 6,200), which represented > 15% of the total population of GAD+ cells in the basal forebrain (estimated at approximately 39,000), were retrogradely labeled from the LHp. These cells were distributed through the basal forebrain cell groups, where ChAT+ cells are also located, including the medial septum and diagonal band nuclei, the magnocellular preoptic nucleus, and the substantia innominata, with few cells in the globus pallidus. In these same nuclei, a small number of ChAT+ cells were retrogradely labeled (estimated at approximately 800), which represented only a small percentage (< 5%) of the ChAT+ cell population in the basal forebrain (estimated at approximately 18,000). Both the GAD+ and ChAT+ LHp-projecting neurons represented a small subset of their respective populations in the basal forebrain, distinct from the magnocellular, presumed cortically projecting, basal neurons. In addition to the GAD+ cells in the basal forebrain, GAD+ cells in the adjacent preoptic and anterior hypothalamic regions were also retrogradely labeled in significant numbers (estimated at approximately 5,500) and proportion (> 20%) of the total population (estimated at approximately 30,000) from the LHp. The retrogradely labeled GAD+ neurons were distributed in continuity with those in the basal forebrain through the lateral preoptic area, medial preoptic area, bed nucleus of the stria terminals, and anterior and dorsal hypothalamic areas. Of the large number of cells that project to the LHp in the basal forebrain and preoptic-anterior hypothalamic regions (estimated at approximately 66,000), the GAD+ neurons represented a significant proportion (> 15%) and the ChAT+ neurons a very small proportion (approximately 2%). The relative magnitude of the GABAergic projection suggests that it may represent an important inhibitory influence of the descending efferent output from the basal forebrain and preoptic-anterior hypothalamic regions.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAergic and other noncholinergic basal forebrain neurons, together with cholinergic neurons, project to the mesocortex and isocortex in the rat.

The extrathalamic relay from the brainstem reticular formation to the cerebral cortex in the basal forebrain has been thought to be constituted predominantly, if not exclusively, by cholinergic neurons. In contrast, the septohippocampal projection has been shown to contain an important contingent of gamma-aminobutyric acid (GABA)ergic neurons. In the present study, we investigated whether GABAergic neurons also contribute to the projection from the basal forebrain to neocortical regions, including the mesocortex (limbic) and the isocortex in the rat. For this purpose, retrograde transport of cholera toxin (CT) was examined from the medial prefrontal cortex for the mesocortex and from the parietal cortex for the isocortex and was combined with dual-immunohistochemical staining for either choline acetyltransferase (ChAT) or glutamic acid decarboxylase (GAD) in adjacent series of sections. Retrogradely labelled GAD+ neurons were codistributed with retrogradely labelled ChAT+ neurons through the basal forebrain from both the prefrontal and the parietal cortex, suggesting parallel, widespread cortical projections. The GAD+ cortically projecting cells were similar in size to the ChAT+ cells, thereby indicating that they comprise a contingent of the magnocellular basal cell complex. The proportions of retrogradely labelled neurons that were GAD+ (approximately one-third) were equal to or greater than those that were ChAT+ from both the prefrontal cortex and the parietal cortex. In addition, the total of GAD+ and ChAT+ neurons did not account for the total number of cortically projecting cells, indicating that another equivalent proportion of chemically unidentified noncholinergic neurons also contributes to the basalocortical projection. Accordingly, as in the allocortex, GABAergic, cholinergic, and other unidentified noncholinergic neurons may have the capacity to modulate activity in the mesocortex (limbic) and the isocortex through parallel, widespread projections.

Decreased high density lipoprotein-cholesterol levels in male patients with transient ischemic attacks.

Plasma lipid and lipoproteins levels were determined in a continuous series of 50 patients (36 males and 14 females), mean age around 50 years, with a clinical diagnosis of transient ischemic attacks (TIAs). TIA was defined as a sudden episode of focal cerebrovascular insufficiency, with complete resolution of the symptoms within 24 h. TIAs are considered an important prognostic symptom for ischemic cerebrovascular diseases, being manifest in approximately 45% of the patients later undergoing a complete stroke. Plasma total cholesterol levels did not differ in these patients, when compared with a similar series of patients of the same age and sex, free of cerebrovascular lesions. A slight elevation of mean triglyceride levels was detected in the patients of both sexes, as well as higher incidence of type IV hyperlipoproteinemia. The most significant finding, however, observed only in male TIA patients, was that of significantly reduced high density lipoprotein (HDL)-cholesterol levels. This reduction (-19.7% compared to the control group) is similar to that recently reported for patients with clear-cut ischemic cerebrovascular disease. The detection of decreased HDL-cholesterol levels in male TIA patients may be of considerable significance for a prognostic evaluation of this biochemical parameter.

Binding sites for [3H]2-oxo-quazepam in the brain of the cat: evidence for heterogeneity of benzodiazepine recognition sites.

In the present study, the distribution of benzodiazepine recognition site subtypes in the brain of the cat was investigated. To this aim, the binding properties of [3H]2-oxo-quazepam ([3H]2OXOQ) and [3H]beta-CCE, two ligands with preferential affinity for Type I benzodiazepine recognition sites, were compared to binding parameters for [3H]flunitrazepam ([3H]FNT) in different areas of the cat brain. The ratio of [3H]2OXOQ to [3H]FNT binding sites indicated that, in the cerebellum, Type I sites accounted for 90% of the total number of benzodiazepine recognition sites. The cerebral cortex, thalamus and mesencephalic reticular formation had also a high proportion of Type I sites (73-78%), whilst the two subtypes were almost equally distributed in the hippocampus, amygdala and bulbar reticular formation. A similar distribution of subtypes of benzodiazepine recognition sites was indicated by the ratio of [3H]beta CCE to [3H]FNT binding sites for different areas of the brain. These results demonstrate the existence of heterogeneity of recognition sites for benzodiazepines in the brain of the cat and support the view that [3H]2OXOQ preferentially labels Type I sites.

Enhancement of tonic and phasic events of rapid eye movement sleep following bilateral ibotenic acid injections into centralis lateralis thalamic nucleus of cats.

The excitotoxin ibotenic acid (1.2-2.6 microliters of 50 micrograms/microliters) was injected bilaterally into the thalamic centralis lateralis nucleus of chronically implanted cats in order to study the effects of tonic excitation followed by destruction of perikarya on the sleep-waking cycle and its electrographic correlates. Ibotenate injections were performed under mild ketamine anaesthesia. Immediately afterwards, the animals showed behavioural arousal accompanied first by ocular nystagmiform movements and then by pontogeniculooccipital waves. By 6-10 h post-injection, the numbers of rapid eye movement sleep episodes, but not their duration, increased compared to the preinjection control period. The injection sites were histologically confirmed using conventional Thionin stains. Additional control was provided by retrograde transport of wheat-germ agglutinin conjugated with horseradish peroxidase. The present results suggest that a population of neurons important for ocular saccades, pontogeniculooccipital waves, and the state of desynchronized sleep is present in the internal medullary lamina, in particular in the centralis lateralis nuclei.

GABAergic and cholinergic basal forebrain and preoptic-anterior hypothalamic projections to the mediodorsal nucleus of the thalamus in the cat.

The present study examined projections of GABAergic and cholinergic neurons from the basal forebrain and preoptic-anterior hypothalamus to the "intermediate" part of the mediodorsal nucleus of the thalamus. Retrograde transport from this region of the mediodorsal nucleus was investigated using horseradish peroxidase-conjugated wheatgerm agglutinin in combination with peroxidase-antiperoxidase immunohistochemical staining for glutamic acid decarboxylase and choline acetyltransferase. A relatively large number of retrogradely-labelled glutamic acid decarboxylase-positive neurons are located in the basal forebrain, amounting to more than 7% of the total population of glutamic acid decarboxylase-positive cells in this region. Moreover, retrogradely-labelled choline acetyltransferase-positive cells are interspersed among glutamic acid decarboxylase-positive neurons, accounting for about 6% of the total choline acetyltransferase-positive cell population in the basal forebrain. The glutamic acid decarboxylase-positive and choline acetyltransferase-positive retrogradely-labelled neurons are distributed throughout several regions of the basal forebrain, including the medial septum, the diagonal band of Broca, the magnocellular preoptic nucleus, the substantia innominata pars anterior, the substantia innominata pars posterior, and the globus pallidus where only a few retrogradely-labelled neurons were seen. The choline acetyltransferase-positive mediodorsal-projecting neurons are morphologically different from the choline acetyltransferase-positive neurons in the basal forebrain, suggesting that those projecting to the mediodorsal nucleus are a small proportion of the cholinergic neuronal population in the basal forebrain. In the preoptic-anterior hypothalamus, many retrogradely-labelled glutamic acid decarboxylase-positive cells were found, amounting to more than 7% of the total population of glutamic acid decarboxylase-positive cells in this region. These retrogradely-labelled glutamic acid decarboxylase-positive neurons are distributed throughout the preoptic-anterior hypothalamus in a continuous line with those in the basal forebrain, including the lateral preoptic area, the medial preoptic area, the bed nucleus of the stria terminalis, and the anterior and dorsal hypothalamic areas. The highest percentage of mediodorsal-projecting GABAergic neurons is in the anterior lateral hypothalamus where more than 25% of the total population of glutamic acid decarboxylase-positive cells project to the mediodorsal nucleus of the thalamus. Overall, of the large population of retrogradely-labelled neurons in the basal forebrain and preoptic-anterior hypothalamus, a significant proportion are glutamic acid decarboxylase-positive neurons (> 60% in the basal forebrain and > 30% in the preoptic-anterior hypothalamus), while the choline acetyltransferase-positive neurons amount to a smaller percentage of the neurons projecting to the mediodorsal nucleus (< 13% in the basal forebrain and < 2% in the preoptic-anterior hypothalamus). These results provide anatomical evidence of direct GABAergic projections from the basal forebrain and preoptic-anterior hypothalamic regions to the "intermediate" part of the mediodorsal nucleus in the cat. This GABAergic projection field could be the direct pathway by which the basal forebrain directly modulates thalamic excitability and may also be involved in mechanisms modulating electroencephalographic synchronization and sleep through the "intermediate" mediodorsal nucleus.

Calcitonin binding site distribution in the cat central nervous system: a wider insight of the peptide involvement in brain functions.

Calcitonin (CT) binding site distribution has been studied in the cat CNS. The autoradiographic analyses of [125I]-eelCT (ECT) binding showed high density of silver grains in the mesencephalic PAG, in the raphe nuclei and in the dorsal horns, laminae I, IV, V, and VI, where ECT may act to inhibit nociceptive transmission. Other binding-rich areas included the caudatus, the amygdala, the hypothalamus, the substantia nigra, the locus coeruleus and the formatio reticularis mesencephalica. Medium to low density was seen, amongst other areas in the cortex piriformis, the hippocampus, the medial and intralaminar thalamus and the tractus spino-thalamicus. ECT binding site distribution revealed essentially homologous locations in the cat and rat CNS. At difference, the presence of binding in the piriform cortex and in discrete thalamic nuclei suggests a widespread involvement of ECT in a variety of central functions in addition to what already demonstrated.

Stress-related changes in calcitonin gene-related peptide binding sites in the cat central nervous system.

The possibility of area-specific changes in binding sites for CGRP in response to stress was studied in cat CNS after repeated sleep-deprivation and restriction of movement. Brain sections were obtained from a cat placed under stressful conditions for 2 h the 1st day, 6 h the 2nd day and 24 h the 3rd day. Changes in CGRP binding sites were evaluated by an in vitro autoradiographic technique with 125I-Tyr-rat-CGRP as a ligand. The autoradiograms were then compared with those of control animals. The results show decreased labelling in the cortex prefrontalis and pyriformis and in some basal ganglia (n. caudatus, claustrum, n. entopedencularis). Increased CGRP binding site densities were seen in areas involved in the integration of sensory information, in the control of endocrine secretion and in those that participate in sleep-walking cycles. These changes in CGRP binding in selective CNS areas following stress suggests that CGRP plays a role in processes of adaptation.

Changes in EEG spindle activity induced by ibotenic acid lesions of medialis dorsalis thalamic nuclei in the cat.

The injection of an excitotoxin into medialis dorsalis thalamic nuclei (MD) elicited a short-term increase followed by a depression on EEG spindle waves in chronically implanted cats. This biphasic action provides further evidence to the hypothesis that MD plays a crucial role in transferring and inducing spindling on frontal cortex. In addition, retrograde horseradish peroxidase transport from previously lesioned MD labeled subcortical structures such as basal forebrain, anterior hypothalamus, reticular thalamic nucleus, ventral tegmental area, and locus coeruleus.

Limbic and brainstem afferents to thalamic mediodorsal nucleus: a horseradish peroxidase study.

Subcortical ipsilateral afferents to mediodorsal (MD) thalamic nucleus were investigated by retrograde transport of horseradish peroxidase (HRP). HRP was injected into the rostral and caudal part of MD following both a vertical and an oblique stereotaxic approach. We have identified MD afferents from the following structures: area septalis, amygdaloid complex, hypothalamus, mamillary bodies, tuberculum olfactorium, claustrum, ventral tegmental area, zona incerta, substantia nigra, griseum centralis, formatio reticularis mesencephali and pontis oralis. The projections from hypothalamus, amygdaloid nuclei and area septalis and reticular formation are in agreement with the integrative function of sensory afferents of this thalamic nucleus.

Ibotenic acid lesions of the thalamic medialis dorsalis nucleus in the cat: effects on the sleep-waking cycle.

Bilateral ibotenic acid lesions of the thalamic medialis dorsalis nucleus in chronically implanted cats produced a significant reduction of slow wave sleep. Rapid eye movement sleep was also affected but in a less significant manner. The lesions were limited to the intermediate portion of the nucleus, which receives projections from the brainstem and prosencephalic structures implicated in sleep mechanisms. These results are consistent with the hypothesis that the medialis dorsalis nucleus plays an important role in the induction and maintenance of sleep.

Motoneurone recurrent inhibition is enhanced by L-acetylcarnitine in humans.

Renshaw cells, which mediate the recurrent inhibition of spinal a-motoneurones, are activated by acetylcholine, both through motoneurone collaterals and the reticulo-spinal system. Since it is known that L-acetylcarnitine (L-AC) has central cholinergic effects, we tested in ten normal subjects and three spastic patients the ability of L-AC to induce changes in excitability of the Renshaw cells. These were activated by a conditioning monosynaptic reflex of the soleus muscle, evoked by electrical stimulation of the tibial nerve, and the resulting recurrent inhibition of the motoneurones was assessed by a subsequent monosynaptic reflex (H'). Recurrent inhibition was tested prior to, during and after an intravenous administration of a solution containing 2000 mg of L-AC. L-AC administration proved to be able to induce in all subjects a decrease in the H'-reflex. This effect ensued approximately 30 min after onset of L-AC administration, reached the peak after 40 min and vanished in about one hour. The extent of the decrease in H' varied among subjects, being on the average 22% of the control values. A relationship was found between duration of L-AC administration and time for reaching the maximal effect. These results show that L-AC is able to decrease a-motoneurone excitability by increasing Renshaw cell activity, both in normal and spastic subjects.

Short-latency inhibition of soleus motoneurones by impulses in Ia afferents from the gastrocnemius muscle in humans.

1. The possibility that the Ia afferent fibres from the gastrocnemius medialis muscle could be responsible for a decrease in excitability of the soleus motor pool was investigated. 2. The soleus H reflex, evoked by tibial nerve stimulation in the popliteal fossa, was conditioned by a single stimulus to the gastrocnemius medialis nerve at various stimulus intensities and conditioning-test intervals. Care was taken to avoid spread of current from the conditioning stimulus to the tibial nerve, and the results obtained by surface stimulation were compared with those obtained by stimulation through a needle whose tip was positioned closer to the nerve. 3. Stimulation of the gastrocnemius medialis nerve induced two short-lasting periods of inhibition in the soleus H reflex, peaking at about 0 and 5 ms of conditioning-test delay. The early inhibition could begin at a stimulus strength as low as 0.5 x MTh (the Motor Threshold). The later inhibition appeared on greater stimulus strength than the earlier. 4. Prolonged vibration of the Achilles tendon abolished the capability of the conditioning stimulus to induce the short-latency inhibition of the soleus H reflex. 5. By stimulating the gastrocnemius medialis nerve at two points separated by a known distance, the conduction velocity of the fibres responsible for the early inhibition was estimated, and found to be around 100 m s-1. 6. Isometric leg flexion, accomplished by tonic activation of gastrocnemius medialis and lateralis but not soleus, was able to induce an inhibition of the soleus H reflex even at very low levels of gastrocnemius electromyographic activity. 7. These findings strongly suggest the existence of an inhibitory effect of primary spindle afferent fibres from the gastrocnemius medialis muscle onto the soleus motor pool. This is not unexpected, since the gastrocnemius medialis muscle can be either agonist or antagonist to the soleus muscle in the performance of different movements.

Influences of locus ceruleus, raphe dorsalis, and periaqueductal gray matter on somatosensory-recipient thalamic nuclei.

Spontaneous and evoked discharge of neurons in the nucleus ventralis posterolateralis (VPL) and spontaneous discharge of neurons in the posterior group and nucleus lateralis posterior (LP) were conditioned by brief trains of stimuli to the locus ceruleus (LC), raphe dorsalis (RD), and periaqueductal gray matter (PAG) in cats anesthetized with pentobarbital or ketamine. Stimulation of LC and RD was without effect on VPL neurons, but induced a long-latency, long-lasting inhibition of LP neurons. Stimulation of the PAG induced marked inhibition of the firing of neurons in all three thalamic nuclei. No differences were found between cats anesthetized with ketamine or pentobarbital.

Recurrent and reciprocal inhibition of the human monosynaptic reflex shows opposite changes following intravenous administration of acetylcarnitine.

A long-latency, long-lasting increase in the recurrent inhibitory effect on the soleus monosynaptic (Hoffmann, H) reflex was induced after intravenous administration of L-acetylcarnitine, a substance known to process central cholinergic activity. This effect was paralleled by disappearance of the H reflex inhibition (functionally disinhibition) induced by stimulation of Ia afferent fibres from the tibialis anterior (reciprocal inhibition) and gastrocnemius medialis muscle. Magnitude and time course of the L-acetylcarnitine-induced effects were significantly correlated. The data suggest that (1) the L-acetylcarnitine depression of the reciprocal inhibition is mediated by excitation of Renshaw cells impinging on Ia interneurones (INs), and (2) the inhibitory effect of GM Ia afferents onto Sol is mediated by INs subjected to Renshaw inhibition. The results point to the similarity in the wiring of the 'output stage' circuit between cat and humans, and provide a method for testing this network in man.

Convergence of Ia fibres from synergistic and antagonistic muscles onto interneurones inhibitory to soleus in humans.

1. The possibility that Ia afferent fibres from the gastrocnemius medialis (GM) and from the tibialis anterior (TA) muscle could converge on to a single interneuronal pool inhibitory to the soleus motoneurones was investigated. 2. The soleus H reflex, evoked by tibial nerve stimulation in the popliteal fossa, was conditioned by separate or combined stimulation of the nerves to the GM or TA muscles. Stimulus intensity was below the motor threshold (MTh), and the conditioning-test intervals were such as to evoke short-latency inhibition of the soleus H reflex. Care was taken to avoid current spread and artifacts connected with the closeness in time and space of the conditioning and test stimuli. 3. Separate stimulation of both GM and TA nerves was able to induce significant inhibitory effects on the H reflex amplitude at stimulus strengths larger than 0.75 x MTh, on the average. Combined stimulation of the two nerves was able to reduce the H reflex at lower stimulus strengths, at which either nerve was ineffective alone. 4. Conditioning stimulus strengths close to the MTh reduced the H reflex to approximately 80% of the control value, both on single and combined stimulation, i.e. saturation of the inhibitory effect was found. 5. By extrapolating the regression line through the normalized data from all subjects, it was assumed that the smallest stimulus strength necessary to drive the inhibitory interneurones to threshold was, on the average, 0.5 and 0.6 x MTh, on combined and separate nerve stimulation, respectively. 6. Tonic voluntary activation of the soleus abolished the inhibitory effects of both separate and combined stimulations. This was tested on the H reflex, on the rectified and averaged EMG, and on the peristimulus histogram of single motor unit discharge. 7. The findings strongly suggest the existence of spatial summation of the effects from GM and TA muscle at the level of a single interneuronal pool. Most probably, the responsible afferent fibres are group I spindle afferents, and the interneurones those mediating the reciprocal inhibition. The data do not support the notion of parallel pathways, exclusive to each nerve.

The synchronizing influence of Substantia Innominata on the thalamus of the cat.

We examined the stimulating effect of Substantia Innominata pars anterior (SIa), during the waking state, on the 'central' part of the Mediodorsal nucleus of the thalamus (MD), combining electrophysiological and anatomical techniques in restrained, undrugged, unanaesthetized cats. Thalamic MD units were recorded, after electrical stimulation of the Substantia Innominata, at 1 Hz, with a single pulse or short trains of four pulses. Responses were studied by poststimulus histograms. In about 64 of the 84 recorded MD neurones (76%), stimulation of the Substantia Innominata, during the waking state, induced a brief cell excitation, followed first by prolonged inhibition of firing and then by a strong excitatory rebound discharge; after this comes a second sequence of inhibition and excitation, of decreasing amplitude. After stimulation of the Substantia Innominata, the MD units tended to start a repetitive discharge at 4--7 Hz. To investigate the connections of Substantia Innominata cells upon the areas where MD units were recorded we injected horseradish peroxidase wheat germ agglutinin (WGA-HRP), combined with immunohistochemistry for glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT). Of the total population of retrogradely labelled cells in the Substantia Innominata 53% were GAD positive while less than 16% were ChAT positive. The GAD positive MD-projecting cells in the Substantia Innominata were triangular to fusiform and small to medium in size. These findings indicate that GABAergic input from the Substantia Innominata may contribute to increasing the hyperpolarizing inhibitory pressure on MD cells in the 'central' part during slow wave sleep (SWS).

Pepsinogens: physiology, pharmacology pathophysiology and exercise.

Human gastric mucosa contains aspartic proteinases that can be separated electrophoretically on the basis of their physical properties into two major groups: Pepsinogen I (PGA, PGI); and Pepsinogen II (PGC, PGII). Pepsinogens consist of a single polypeptide chain with molecular weight of approximately 42,000 Da. Pepsinogens are mainly synthesized and secreted by the gastric chief cells of the human stomach before being converted into the proteolytic enzyme pepsin, which is crucial for the digestive processes in the stomach. Pepsinogen synthesis and secretion are regulated by positive and negative feed-back mechanisms. In the resting state pepsinogens are stored in granules, which inhibit further synthesis. After appropriate physiological or external chemical stimuli, pepsinogens are secreted in the stomach lumen where hydrochloric acid, secreted by the parietal cells, converts them into the corresponding active enzyme pepsins. The stimulus-secreting coupling mechanisms of pepsinogens appear to include at least two major pathways: one involving cAMP as a mediator, the other involving modification of intracellular Ca(2+)concentration. Physiological or external chemical stimuli acting through the intracellular metabolic adenyl cyclase are more effective in inducing ' de novo ' pepsinogen synthesis than those acting through intracellular Ca(2+). The activation of protein kinase C (PK-C) would appear to be involved in regulatory processes. The measurement of pepsinogens A and C in the serum is considered to be one of the non-invasive biochemical markers for monitoring peptic secretion and obtaining information on the gastric mucosa status of healthy subjects. Recently, pepsinogen measurements have been used as an effective biochemical method for evaluating and monitoring patients with gastrointestinal diseases and for checking the effects of drug treatment. The level of PGA in the serum is always high in normal gastritis, while in atrophic gastritis it is always low. In both cases the PGC level in the serum is high. In most gastrointestinal pathologies the ratio between the PGA/PGC decreases. Various reports concerning hormone and/or enzyme modification as well as gastrointestinal distress in the case of long distance exercise have been reported. It has been suggested that the origin of the gastrointestinal distress experienced by long distance runners is a transient ischaemia of the gastric mucosa; it is also suggested that a hypobaric-hypoxic environment could contribute to induce gastric mucosa necrosis. Interrelation between gastrointestinal distress, hypobaric-hypoxic environment and modifications of PGA and PGC, gastrin and cortisol was evaluated in 13 athletes after a marathon performed at 4300 m. Gastrointestinal symptoms occurred in approximately 40% of the athletes. After the race the athletes showed a significant increase of gastrin and cortisol, while the ratio between PGA/PGC decreased. No relationship was observed between gastrointestinal symptoms and hormonal changes after the race. A control group of five subjects, who had been exposed to the same environmental conditions, showed no gastrointestinal or hormonal alteration. Conversely, control subjects presented a significant decrease of cortisol related to the circadian rhythm. The same incidence of gastrointestinal symptoms at high altitude and at sea level and the absence of pathological alteration of PGA and PGC in the serum of the athletes indicates that running a marathon and living for 6 days at 4300 m does not induce gastric mucosa necrosis. Cortisol and gastrin alteration observed in the athletes at this altitude would seem to be related to an activation of the mesopontine and forebrain structures involved in the behavioural and metabolic integration of the autonomic control and arousal and psychophysical-exercise stress. 2000 Academic Press@p$hr