Circling behavior elicited from the pedunculopontine nucleus: evidence for the involvement of hindbrain GABAergic projections

Unilateral microinjection of GABA agonists into the pedunculopontine nucleus (PPN) of the rat resulted in contraversive postural asymmetry and circling behavior; GABA antagonist caused ipsiversive asymmetry and circling when applied to the PPN. A hemitransection was placed immediately caudal to substania nigra (SN) and rostral to PPN in order to interrupt all connections between the PPN and ipsilateral forebrain nuclei. After hemitransection, microinjection of GABAergic drugs into the PPN on the hemitransected side produced postural asymmetry and circling identical to that observed in intact rats. The hemitransection resulted in a loss of glutamic acid decarboxylase activity in PPN (25%) not substantially greater than that observed in animals with unilateral destruction of SN, indicating that a major proportion of GABA terminals in PPN are derived from hindbrain sources. It appears that forebrain (that is, nigrotegmental) GABAergic projections are not essential for the GABA-mediated asymmetry elicited from PPN.     

GABA antagonists applied to the ventral surface of the medulla oblongata block the baroreceptor reflex

Application of gamma-aminobutyric acid (GABA) antagonist drugs (bicuculline, picrotoxinin) to the intermediate region of the ventral surface of the medulla oblongata of gallamine paralyzed, alpha-chloralose anesthetized cats produced a dose-dependent blockade of the baroreceptor reflex. GABA antagonists applied to the rostral and caudal areas did not block the baroreceptor reflex. When strychnine was applied to the ventral medulla, it did not block the baroreceptor reflex. The diffusion of [3H]bicuculline was studied by scintillation counting and autoradiography and found not to penetrate more than 2 mm from the ventral medullary surface. These results suggest that a GABAergic synapse lies within the first 2 mm of the ventral medulla and is involved in baroreceptor modulation of the sympathetic outflow.     

Evidence that bicuculline and picrotoxin act at separate sites to antagonize γ-aminobutyric acid in rat cuneate nucleus

Bicuculline and picrotoxin have been compared over a wide range of concentrations as antagonists of γ-aminobutyric acid (GABA) and muscimol. Both antagonists caused an approximately parallel displacement of the agonist dose-response curves constructed from depolarizing responses of afferent nerve fibres in the cuneate nucleus slice. A Schild plot of log (muscimol dose ratio ? 1) vs log (bicuculline concentration) had a slope of 0.070 ± 0.032 while a similar plot for picrotoxin had a slope of 0.635 ± 0.021. When GABA was used as the agonist, the Schild plots for both antagonists had significantly lower slopes, probably due to the influence of GABA uptake processes. The degree of antagonism obtained with combinations of bicuculline and picrotoxin indicated that these two antagonists were acting at independent sites. It is concluded that bicuculline antagonizes GABA and muscimol competitively at the receptor whereas picrotoxin acts at some stage in the depolarization response mechanism.     

Albumin extravasation in bicuculline-induced blood-brain barrier dysfunction

The extravasation of endogeneous rat albumin and exogeneous ¹²⁵I-labeled human serum albumin was compared in rats subjected to bicuculline-induced blood-brain barrier dysfunction.
The correlation between rocket immunoelectrophoretic assays of endogeneous rat albumin and ¹²⁵I-labeled human serum albumin, assayed by gamma scintillation counting, was good irrespective of whether ¹²⁵I-labeled albumin was studied in whole brain tissue or in brain homogenates. The ratio of brain to serum albumin was similar with the two assay methods.     

Medullary ventral surface GABA receptors affect respiratory and cardiovascular function

We previously demonstrated that GABA and muscimol administered either into the cisterna magna or the fourth ventricle to chloralose-anesthetized cats cause respiratory depression, hypotension, and bradycardia. Injection of these substances into the lateral and third ventricles had no effect. In order to localize the site of action, muscimol and GABA were applied by Perspex rings to the ventral surface of the medulla. Application of muscimol (0.25-2.66 micrograms) to Schlaefke's area in 6 cats reduced minute ventilation from 443 +/- 38 to 291 +/- 52 ml/min by reducing tidal volume from 31.8 +/- 2.3 to 17.6 +/- 1.4 ml, without changing respiratory rate and duration of inspiration. Hypotension and bradycardia were also observed. Application of GABA (0.14-4.86 mg) produced similar effects on respiratory activity and arterial blood pressure. No significant effects occurred when high doses of these agents were applied to Loeschcke's and Mitchell's areas. Application of bicuculline (5-25 micrograms) to Schlaefke's area had the opposite effect of muscimol and GABA on respiratory activity and blood pressure, and reversed the respiratory and cardiovascular depressant effects of both agents. We conclude that GABA receptors are present at Schlaefke's area, and that activation of these receptors results in respiratory depression, hypotension, and bradycardia. Our results suggest that GABA may be an important inhibitory neurotransmitter in the modulation of respiratory and cardiovascular control.     

INFLUENCE OF INTRACEREBROVENTRICULAR ADMINISTRATION OF TETANUS TOXIN ON EXPERIMENTAL SEIZURES AND PROTECTION AFFORDED BY SOME ANTIEPILEPTIC DRUGS IN MICE

The dose of the intracerebroventricularly administered (i.c.v.) tetanus toxin (Tetx) evoking the death of 50% of experimental mice (LD₅₀) was estimated to be 18.0 (11.5–28.2) times the minimal lethal dose (MLD). MLD is defined as the lowest dose of Tetx necessary to kill a 20-g albino mouse within 96 h after intraperitoneal treatment. Tetx (0.25 and 0.5 LD₅₀) increased the convulsive threshold of electric current from 24 to 96 and 120 h, respectively, following i.c.v. administration. Both doses of Tetx diminished convulsant potencies of pentylenetetrazole, bicuculline, aminophylline and pilocarpine 24 h after application. At the same time Tetx (0.5 LD₅₀) increased the protection afforded by carbamazepine, valproate, phenobarbital and diazepam in maximal electroshock (MES) by approximately 36, 11, 21 and 26%, respectively, without affecting total blood plasma levels of antiepileptic drugs. No marked changes in γ-aminobutyric acid (GABA) concentration and total activity ofl-glutamic acid decarboxylase (GAD) assessed in the whole-brain homogenates resulted from Tetx treatment. Our results seem to indicate that low doses (<LD₅₀) of i.c.v. administered Tetx may lead to a relative prevalence of inhibitory over excitatory processes in the central nervous system suggesting a complex action of Tetx at the neuronal level.     

GABAergic mechanisms in regulating the activity state of substantia nigra pars reticulata neurons

Substantia nigra reticulata is the major output structure of the basal ganglia involved in somatosensory integration and organization of movement. While previous work in vitro and in anesthetized animal preparations suggests that these neurons are autoactive and points to GABA as a primary input regulating their activity, single-unit recording coupled with iontophoresis was used in awake, unrestrained rats to further clarify the role of tonic and phasic GABA input in maintenance and fluctuations of substantia nigra reticulata neuronal activity under physiologically relevant conditions. In contrast to glutamate, which was virtually ineffective at stimulating substantia nigra reticulata neurons in awake rats, all substantia nigra reticulata neurons tested were inhibited by iontophoretic GABA and strongly excited by bicuculline, a GABA-A receptor blocker. The GABA-induced inhibition had short onset and offset latencies, a fading response pattern (a rapid decrease in rate followed by its relative restoration), and was independent of basal discharge rate. The bicuculline-induced excitation was inversely related to discharge rate and current (dose)-dependent in individual units. However, the average discharge rate during bicuculline applications at different currents increased to a similar plateau (60 impulses/s), which was about twice the mean basal rates. The excitatory effects of bicuculline were phasically inhibited or completely blocked by brief GABA applications and generally mimicked by gabazine, another selective GABA antagonist. These data as well as neuronal inhibitions induced by nipecotic acid, a selective GABA uptake inhibitor, suggest that substantia nigra reticulata neurons in awake, quietly resting conditions are under tonic, GABA-mediated inhibition.

Therefore, because of inherent autoactivity and specifics of afferent inputs, substantia nigra reticulata neurons are very sensitive to phasic alterations in GABA input, which appears to be the primary factor determining fluctuations in their activity states under physiological conditions. While these cells are relatively insensitive to direct activation by glutamate, and resistant to a continuous increase in GABA input, they appear to be very sensitive to a diminished GABA input, which may release them from tonic inhibition and determine their functional hyperactivity.     

Muscimol microinjection into cerebellar fastigial nucleus exacerbates stress-induced gastric mucosal damage in rats

Aim:

To investigate the effects of microinjection of the GABA_A receptor agonist muscimol into cerebellar fastigial nucleus (FN) on stress-induced gastric mucosal damage and the underlying mechanism in rats.

Methods:

Stress-induced gastric mucosal damage was induced in adult male SD rats by restraining and immersing them in cold water for 3 h. GABA_A receptor agonist or antagonist was microinjected into the lateral FN. The decussation of superior cerebellar peduncle (DSCP) was electrically destroyed and the lateral hypothalamic area (LHA) was chemically ablated by microinjection of kainic acid. The pathological changes in the gastric mucosa were evaluated using TUNEL staining, immunohistochemistry staining and Western blotting.

Results:

Microinjection of muscimol (1.25, 2.5, and 5.0 μg) into FN significantly exacerbated the stress-induced gastric mucosal damage in a dose-dependent manner, whereas microinjection of GABA_A receptor antagonist bicuculline attenuated the damage. The intensifying effect of muscimol on gastric mucosal damage was abolished by electrical lesion of DSCP or chemical ablation of LHA performed 3 d before microinjection of muscimol. Microinjection of muscimol markedly increased the discharge frequency of the greater splanchnic nerve, significantly increased the gastric acid volume and acidity, and further reduced the gastric mucosal blood flow. In the gastric mucosa, further reduced proliferation cells, enhanced apoptosis, and decreased anti-oxidant levels were observed following microinjection of muscimol.

Conclusion:

Cerebellar FN participates in the regulation of stress-induced gastric mucosal damage, and cerebello-hypothalamic circuits contribute to the process.     

GABA-mediated inhibition correlates with orientation selectivity in primary visual cortex of cat

Orientation selectivity is an important emergent property of neurons in the primary visual cortex, and inhibition is thought to play an important role in establishing this selectivity. But the relationship between strength of inhibition and orientation selectivity is unclear. To investigate this relationship, we electrophoretically applied the inhibitory transmitter GABA and the GABA(A) antagonist bicuculline on the same individual area 17 neurons in anesthetized cats. Neurons were classified as weakly orientation-selective, moderately orientation-selective, or strongly orientation-selective, according to the values of an orientation bias index. Orientation bias, half-width of the tuning curve at half-height and an orientation-specificity index (orthogonal to optimal ratio) were compared with or without GABA and bicuculline administration. GABA improved orientation selectivity with the greatest effects on weakly orientation-selective cells, smaller effects on moderately orientation-selective cells, and minimal effects on strongly orientation-selective cells; bicuculline diminished orientation selectivity the most on moderately and strongly orientation-selective cells, with minimal effects on weakly orientation-selective cells. We also found that orientation selectivity correlated with the level of neurons' spontaneous activity. These results suggest that the degree of orientation selectivity of an area 17 neuron correlates with its endogenous inhibition strength, and that GABAergic inhibition can bi-directionally regulate orientation selectivity. This correlation indicates that GABA-mediated inhibition plays an important role in establishing sharp orientation selectivity of individual neurons.     

Bicuculline-sensitive gamma-aminobutyrate binding processes in a synaptosome-enriched fraction of rat cerebral cortex.

The binding of low concentrations of [³H]y-aminobutyrate (GABA) to a synaptosomal fraction of rat cerebral, cortex was studied in the presence and absence of added Na⁺; unlabelled GABA and bicuculline methiodide were used to estimate ‘GABA-sensitive’ and ‘bicuculline-sensitive’ binding sites. Significant amounts of [³H]GABA were bound to the particles in both the presence and absence of added Na⁺. In Na⁺-free medium, a single bicuculline-sensitive, high-affinity [³H]GABA binding process was detected, its GABA-sensitive sites having K_B ～- 10^?5M. In Na⁺-containing medium, two bicuculline-sensitive, high-affinity [³H]GABA binding processes were detected, the lower-affinity process having GABA-sensitive sites with K_B ～- 10^?5M and the higher-affinity process having GABA-sensitive sites with K_B ～- 4 × 10^?8MOur findings suggest that bicuculline-sensitive components of [³H]GABA binding in the absence of Na⁺ and in the presence of Na⁺ (higher-affinity process) could represent estimates of Na⁺-indepen dent and Na⁺-dependent synaptic GABA-receptors. The bicuculline-sensitive component of lower-affinity [³H]GABA binding in the presence of Na⁺ might be related to an action of bicuculline on GABA transport. Two GABA-receptor agonists displaced about the same amount of [⁺H]GABA as did bicuculline in Na⁺-free medium, indicating further that a synaptic GABA-receptor was involved in [³H]GABA binding under these conditions. Nipecotic acid markedly inhibited [³H]GABA binding in both the presence and absence of added Na⁺, indicating that some transport sites become occupied by GABA in the absence of added Na⁺ and that nipecotic acid might interact to some extent with synaptic GABA-receptors.