Proliferative activity in incubated slices of immature rat cerebellum

Slices of 8-day-old rat cerebella were prepared with a tissue chopper, incubated in an oxygenated Krebs-Henseleit solution followed by processing for histology. One micrometer thick sections of resin-embedded slices were stained with Toluidine blue. The external granular layer was shown to maintain its normal proliferative activity up to 3 h of incubation as suggested by the presence of all mitotic phases within this period and by a roughly 50% increase in mitotic index upon a 90-min exposure of incubated slices to 20 microM vinblastine. On this basis the incubated slice of the immature rat cerebellum may serve as a simple tool for the rapid histological testing of short-term mitogenic or cytostatic effects and the vulnerability of a brain germinal layer.     

GABAergic signaling in the developing cerebellum

In the adult central nervous system (CNS), GABA is a predominant inhibitory neurotransmitter that regulates glutamatergic activity. Recent studies have revealed that GABA serves as an excitatory transmitter in the immature CNS and acts as a trophic factor for brain development. Furthermore, synaptic transmission by GABA is also involved in the expression of higher brain functions, such as memory, learning and anxiety. These results indicate that GABA plays various roles in the expression of brain functions and GABAergic roles change developmentally in accordance with alterations in GABAergic transmission and signaling. We have investigated morphologically the developmental changes in the GABAergic transmission system and the key factors important for the formation of GABAergic synapses and networks using the mouse cerebellum, which provides an ideal system for the investigation of brain development. Here, we focus on GABA and GABA(A) receptors in the developing cerebellum and address the processes of how GABA exerts its effect on developing neurons and the mechanisms underlying the formation of functional GABAergic synapses.     

Age-dependent and selective binding of beta-bungarotoxin to GABAergic neurons in the rat cerebellum

beta-Bungarotoxin (BuTx)-binding cells were immunocytochemically examined in the developing rat cerebellum. The tissue was incubated with BuTx and then immunostained with antiserum against its toxoid. On postnatal day 6, only Golgi cells were positive for immunoreaction. Immunoreactive Golgi cells were reduced in number on day 15 and disappeared on day 25. On day 15, Purkinje cells were strongly stained, while some basket and stellate cells stained weakly. On day 25 and in adult, basket and stellate cells were more immunoreactive than Purkinje cells. Thus, age-dependent and selective binding of BuTx was restricted to gamma-aminobutyric acid (GABA)ergic neurons.     

Glutamate dehydrogenase in aminoacidergic structures of the postnatally developing rat cerebellum

In the cerebellar cortex of the rat, histochemically demonstrable activity of glutamate dehydrogenase (GDH) was found to increase markedly after the second week of postnatal life. As evaluated histophotometrically, the amount of reaction product in Purkinje cell perikarya exceeded on day 40 that on day 5 by about 85%, whereas the intensity of GDH staining in granule cell bodies and in the molecular layer rose from postnatal day 5 to day 40 up to 325% and 400%, respectively. Due to the parallelism with the onset of aminoacidergic transmission processes, the results are interpreted as indicating the participation of GDH in the metabolism both of transmitter glutamate and of gamma-aminobutyric acid (GABA).     

New candidates for GABAergic neurons in the rat cerebellum: an immunocytochemical study with anti-GABA antibody

Cerebellar cortical neurons immunoreactive to anti-gamma-aminobutyric acid (GABA) antibody were examined in the rat. In addition to the Purkinje, Golgi, basket and stellate cells, spindle-shaped cells lying just below the Purkinje cell layer were found to be strongly immunoreactive to the antibody. By the combination of immunofluorescence and hematoxylin stainings, these GABA-positive cells were shown to be the Lugaro cells. Unlike the immunopositive small Golgi cell, the pale cell was not immunoreactive to the antibody.     

Adenosine signalling at immature parallel fibre–Purkinje cell synapses in rat cerebellum

The purine adenosine is an extracellular signalling molecule involved in a large number of physiological and pathological conditions throughout the mammalian brain. However little is known about how adenosine release and its subsequent clearance change during brain development. We have combined electrophysiology and microelectrode biosensor measurements to investigate the properties of adenosine signalling at early stages of cerebellar development, when parallel fibre–Purkinje cell synapses have recently been formed (postnatal days 9–12). At this stage of development, we could detect little or no inhibitory A₁ receptor tone in basal conditions and during trains of stimuli. Addition of pharmacological agents, to inhibit adenosine clearance, had only minor effects on synaptic transmission suggesting that under basal conditions, the concentration of adenosine moving in and out of the extracellular space is small. Active adenosine release was stimulated with hypoxia and trains of electrical stimuli. Although hypoxia released significant concentrations of adenosine, the release was delayed and slow. No adenosine release could be detected following electrical stimulation in the molecular layer. In conclusion, at this stage of development, although adenosine receptors and the mechanisms of adenosine clearance are present there is very little adenosine release.     

Epileptiform discharges and a synchronous GABAergic potential induced by 4-aminopyridine in the rat immature hippocampus

Extracellular field potential recordings were performed in the CA3 subfield of hippocampal slices that were obtained from 10- to 30-day-old rats. During perfusion with medium containing the convulsant drug 4-aminopyridine (4-AP, 50 microM) 3 main types of spontaneously occurring potentials were observed. The first one was a short-lasting (duration: 400-1100 ms) potential with a frequency of occurrence that ranged between 0.6 and 1.3 Hz. Thus it resembled an epileptiform interictal event. The second type was reminiscent of an ictal epileptiform discharge, lasted 10-35 s and recurred every 40-100 s. The third one was of opposite polarity as compared with the other two types, occurred every 10-100 s and was often followed by the ictal discharge. When recorded in isolation this potential lasted 1.2-2 s. The interictal and ictal discharges were blocked by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), while the potential of opposite polarity was not affected by this pharmacological procedure. It was, however, blocked in a reversible way by the GABAA receptor antagonist bicuculline methiodide (5 microM). These results indicate that in addition to epileptiform activity of interictal and ictal type, 4-AP also induces in the immature rat hippocampus a synchronous event that is due to the activation of the GABAA receptor.     

Excitatory action of an immature glycinergic/GABAergic sound localization pathway

Most mammals determine the azimuthal direction of incoming sound using auditory cues arising from differences in interaural sound intensity. The first station in the ascending auditory pathway, which processes interaural intensity differences, is the lateral superior olive (LSO), a binaural nucleus in the auditory brainstem. LSO neurons encode interaural intensity differences by integrating excitatory input from the ipsilateral cochlea and inhibitory input from the contralateral cochlea. Both inputs converge on single neurons in a highly organized, frequency-specific manner. The correct development of the precise arrangement of these inputs and their physiological properties depends on neuronal activity. Previous studies have shown that inhibitory, glycinergic/GABAergic inputs to the LSO are transiently depolarizing, and it has been hypothesized that this depolarizing action enables developing inhibitory inputs to act as excitatory inputs. In support of this hypothesis, we recently demonstrated that depolarizing glycinergic/GABAergic inputs can increase the intracellular calcium concentration in immature LSO neurons and elicit action potentials. These results provide support for the notion that the influence of glycinergic/GABAergic synaptic activity on development of the LSO involves calcium-dependent signaling mechanisms.     

Adenosine A1 receptor-mediated presynaptic inhibition of GABAergic transmission in immature rat hippocampal CA1 neurons

In the mechanically dissociated rat hippocampal CA1 neurons with native presynaptic nerve endings, namely "synaptic bouton" preparation, the purinergic modulation of spontaneous GABAergic miniature inhibitory postsynaptic currents (mIPSCs) was investigated using whole-cell recording mode under the voltage-clamp conditions. In immature neurons, adenosine (10 microM) reversibly decreased GABAergic mIPSC frequency without affecting the mean current amplitude. The inhibitory effect of adenosine transmission was completely blocked by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM), a selective Alpha(1) receptor antagonist, and was mimicked by N(6)-cyclopentyladenosine (CPA, 1 microM), a selective Alpha(1) receptor agonist. However, CPA had no effect on GABAergic mIPSC frequency in postnatal 30 day neurons. N-ethylmaleimide (10 microM), a guanosine 5'-triphosphate binding protein uncoupler, and Ca(2+)-free external solution removed the CPA-induced inhibition of mIPSC frequency. K(+) channel blockers, 4-aminopyridine (100 microM) and Ba(2+) (1 mM), had no effect on the inhibitory effect of CPA on GABAergic mIPSC frequency. Stimulation of adenylyl cyclase with forskolin (10 microM) prevented the CPA action on GABAergic mIPSC frequency. Rp-cAMPS (100 microM), a selective PKA inhibitor, also blocked the CPA action. It was concluded that the activation of presynaptic Alpha(1) receptors modulates the probability of spontaneous GABA release via cAMP- and protein kinase A dependent pathway. This Alpha(1) receptor-mediated modulation of GABAergic transmission may play an important role in the regulation of excitability of immature hippocampal CA1 neurons.     

Prenatal nicotine exposure alters glycinergic and GABAergic control of respiratory frequency in the neonatal rat brainstem-spinal cord preparation

Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P<0.001). Apnea duration with muscimol averaged 51+/-5.1 s in saline-exposed (N=10), and 86+/-10.6 s in nicotine-exposed (N=12) neonates (P<0.05). These data show that prenatal nicotine exposure alters development of central ventilatory control, and that neurons in the PBC region are involved.     

Glycoprotein transport in the rat cerebellum

Summary The fate of tritiated fucose in rat cerebellum has been followed using autoradiographic techniques for a period up to one week after intraventricular injection. At intervals of up to three hours after injection, Purkinje and granule cell bodies were found to be most heavily labelled. Subsequently this labelling declined and the highest grain density was observed over the molecular layer. The data is interpreted in terms of fucose incorporation into glycoproteins in nerve cell bodies followed by rapid dendritic transport in the Purkinje cell and fast transport in granule cells along ascending axons and parallel fibres.     

缺氧使非洲电鱼小脑浦肯野细胞之间的GABA能突触传递长时程增强

目的:研究急性缺氧对非洲电鱼小脑浦肯野细胞(Pc)之间γ-氨基丁酸(GABA)能突触传递的影响。方法:采用配对全细胞膜片钳记录法,记录电鱼小脑Pc-Pc之间的抑制性突触后电流(IPSC),观察急性缺氧对Pc-Pc IPSC的影响,以及GABA_A受体拮抗剂和谷氨酸α-氨基-3-羟基-5-甲基-4-异噁唑丙酸(AMPA)受体拮抗剂对Pc-Pc IPSC缺氧反应的调节作用。结果:短暂缺氧使Pc-Pc IPSC的幅值显著增大,表现为长时程增强(LTP);GABA_A受体拮抗剂荷包牡丹碱逆转了Pc-Pc IPSC的LTP,表现为长时程抑制;AMPA受体拮抗剂6-氰基-7-硝基喹喔啉-2,3-二酮(CNQX)阻断了Pc-Pc IPSC的LTP,表现为短时程增强。结论:急性缺氧引起电鱼小脑Pc之间的GABA能突触活动持续增强,GABAA受体和AMPA受体共同介导这种反应,提示GABA能和谷氨酸能突触活动的平衡可能是电鱼以及其他缺氧耐受动物缺氧保护反应的关键机制。     

TrkB receptor signaling is required for establishment of GABAergic synapses in the cerebellum

Neurotrophins are essential to the normal development and maintenance of the nervous system. Neurotrophin signaling is mediated by Trk family tyrosine kinases such as TrkA, TrkB and TrkC, as well as by the pan-neurotrophin receptor p75NTR. Here we have deleted the trkB gene in cerebellar precursors by Wnt1-driven Cre--mediated recombination to study the function of the TrkB in the cerebellum. Despite the absence of TrkB, the mature cerebellum of mutant mice appears similar to that of wild type, with all types of cell present in normal numbers and positions. Granule and Purkinje cell dendrites appear normal and the former have typical numbers of excitatory synapses. By contrast, inhibitory interneurons are strongly affected: although present in normal numbers, they express reduced amounts of GABAergic markers and develop reduced numbers of GABAergic boutons and synaptic specializations. Thus, TrkB is essential to the development of GABAergic neurons and regulates synapse formation in addition to its role in the development of axon terminals.     

The GABAergic cerebello-olivary projection in the rat

Summary Immunocytochemical detection of glutamate decarboxylase (GAD), the predominant biosynthetic enzyme of gamma-aminobutyric acid (GABA), reveals the presence of a dense GABAergic innervation in all parts of the inferior olive. One brain center that provides a substantial projection to the inferior olive is the cerebellar nuclei, which contain many small GABAergic neurons. These neurons were tested as a source of GABAergic olivary afferents by combining retrograde tract tracing with GAD immunocytochemistry. As expected from previous studies, injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the inferior olive retrogradely label many small neurons in the interposed and lateral cerebellar nuclei and the dorsal part of the lateral vestibular nucleus, and fewer neurons in the ventro-lateral region of the medial cerebellar nucleus. These projections are predominantly crossed and are topographically arranged. The vast majority, if not all, of these projection neurons are also GAD-positive. The relative contribution of this projection to the GABAergic innervation of the inferior olive was tested by lesion of the cerebellar nuclei, or the superior cerebellar peduncle. Within 10 days the lesion eliminates most GAD-immunoreactive boutons in the principal olive, the rostral lamella of the medial accessory olive, the ventrolateral outgrowth, and the lateral part of the dorsal accessory olive ventral fold. Thus, the effectiveness of this depletion demonstrates that the cerebellar nuclei provide most of the GABAergic innervation to regions of the inferior olive known to receive a cerebellar projection. Moreover, when the lateral vestibular nucleus is damaged, the dorsal fold of the dorsal accessory olive is depleted of GABAergic boutons. The synaptic relations that boutons of the GABAergic cerebello-olivary projection share with olivary neurons were investigated at the electron microscopic level by GAD-immunocytochemistry, anterograde degeneration of the cerebellar axons or anterograde transport of WGA-HRP. All of these methods confirm that GABAergic, cerebello-olivary axon terminals contain pleomorphic vesicles, and synapse on various portions of olivary neurons, and especially on dendritic spines within glomeruli, often in very close proximity to the gap junctions that characteristically couple the dendritic profiles. These results demonstrate four major points: that virtually all of the GABAergic, and presumably inhibitory, neurons of the cerebellar and dorsal lateral vestibular nuclei are projection neurons; that a large portion of the inferior olive receives GABAergic afferents from the cerebellar nuclei; that a portion of the dorsal accessory olive receives GABAergic afferents from the dorsal lateral vestibular nucleus; and that cerebello-olivary fibers often synapse near gap junctions, and therefore could influence electrical coupling of olivary neurons.Abbreviations aMAO subnucleus a of MAO - beta beta nucleus - bMAO subnucleus b of MAO - cMAO subnucleus c of MAO - dc dorsal cap - DC dorsal cochlear nucleus - dfDAO dorsal fold of DAO - dlh dorsal lateral hump of cerebellar nuclei - dIPO dorsal lamella of PO - Gia gigantocellular reticular nucleus - dmcc dorsomedial cell column - GABA gamma-aminobutyric acid - GAD glutamate decarboxylase - HRP horseradish peroxidase - icp inferior cerebellar peduncle - IC inferior colliculus - Inf infracerebellar nucleus - IntA anterior interposed cerebellar nucleus - IntP posterior interposed cerebellar nucleus - Lat lateral cerebellar nucleus - LRt lateral reticular nucleus - LSO lateral superior olive - LVe lateral vestibular nucleus - MAO medial accessory olive - Med medial cerebellar nucleus - Me5 mesencephalic trigeminal nucleus - MVe medial vestibular nucleus - PFl paraflocculus of the cerebellar cortex - PO principle olive - RMg raphe magnus - rMAO rostral lamella of MAO - rs rubrospinal tract - scp superior cerebellar peduncle - SuVe spinal vestibular nucleus - SuVe superior vestibular nucleus - vfDAO ventral fold of DAO - vlo ventrolateral outgrowth - vlPO ventral lamella of PO - Y Y, y vestibular nucleus - WGA wheatgerm agglutinin This paper is dedicated to Professor Fred Walberg on the occasion of his 70th hirthdav     

Isoniazid-induced reduction in GABAergic neurotransmission alters the function of the cerebellar cortical circuit

The cerebellar cortex contributes to the control of movement, coordination, and certain cognitive functions. The cerebellar network is composed of five different types of neurons that are wired together in a repetitive module. Given that four of these five neurons synthesize and release GABA, this inhibitory neurotransmitter plays a central role in regulation of the excitability and correct functioning of the cerebellar cortex. We have now used isoniazid, an inhibitor of glutamic acid decarboxylase, the enzyme responsible for the synthesis of GABA, to evaluate the contribution of GABAergic transmission in different types of cerebellar cortical neurons to the functioning of the cerebellar circuit. Parasagittal cerebellar slices were prepared from 28- to 40-day-old male rats and were subjected to patch-clamp recording in the voltage- or current-clamp mode. Exposure of the tissue slices to isoniazid (10 mM) resulted in a decrease in the level of GABAergic transmission in Purkinje cells and a consequent increase in the firing rate of spontaneous action potentials that was apparent after 40 min. In granule neurons, isoniazid reduced both tonic and phasic GABAergic currents and thereby altered the flow of information across the cerebellar cortex. Our data support the notion that the amount of GABA at the synaptic level is a major determinant of the excitability of the cerebellar cortex, and they suggest that isoniazid may be a useful tool with which to study the function of the cerebellar network.     

Alpha-adrenoceptive dual modulation of inhibitory GABAergic inputs to Purkinje cells in the mouse cerebellum

Noradrenaline (NA) modulates synaptic transmission in various sites of the CNS. In the cerebellar cortex, several studies have revealed that NA enhances inhibitory synaptic transmission by beta-adrenoceptor-and cyclic AMP-dependent pathways. However, the effects of alpha-adrenoceptor activation on cerebellar inhibitory neurotransmission have not yet been fully elucidated. Therefore we investigated the effects of the alpha1- or alpha2-adrenoceptor agonist on inhibitory postsynaptic currents (IPSCs) recorded from mouse Purkinje cells (PCs). We found that the nonselective alpha-adrenoceptor agonist 6-fluoro-norepinephrine increased both the frequency and amplitude of spontaneous IPSCs (sIPSCs). This enhancement was mostly mimicked by the selective alpha1-adrenoceptor agonist phenylephrine (PE). PE also enhanced the amplitude of evoked IPSCs (eIPSCs) and increased the frequency but not the amplitude of miniature IPSCs (mIPSCs). Moreover, PE decreased the paired-pulse ratio of eIPSCs and did not change gamma-aminobutyric acid (GABA) receptor sensitivity in PCs. Conversely, the selective alpha2-adrenoceptor agonist clonidine significantly reduced both the frequency and the amplitude of sIPSCs. Neither eIPSCs nor mIPSCs were affected by clonidine. Furthermore, presynaptic cell-attached recordings showed that spontaneous activity of GABAergic interneurons was enhanced by PE but reduced by clonidine. These results suggest that NA enhances inhibitory neurotransmitter release by alpha1-adrenoceptors, which are expressed in presynaptic terminals and somatodendritic domains, whereas NA suppresses the excitability of interneurons by alpha2-adrenoceptors, which are expressed in presynaptic somatodendritic domains. Thus cerebellar alpha-adrenoceptors play roles in a presynaptic dual modulation of GABAergic inputs from interneurons to PCs, thereby providing a likely mechanism for the fine-tuning of information flow in the cerebellar cortex.     

Dopaminergic innervation and binding in the rat cerebellum

In the present study, we used an antiserum against dopamine (DA), and specific [³H]ligands in order to shed more light on the dopaminergic system of the rat cerebellum. The immunocytochemical approach showed that the entire rat cerebellum is innervated by DA fibers. All cerebellar layers were found to receive a considerable amount of DA afferents but the molecular layer was the most heavily innervated. The analysis of [³H]DA and [³H]spiperone binding showed that in the rat cerebellum there exists DAergic binding with kinetic parameters similar to those reported for the mouse cerebellum. The results of the present study support the existence of a DA system in the rat cerebellum.     

Proliferation of bergmann-glia in the developing rat cerebellum

Summary Mitotic cells in the ganglionic layer of the infant rat cerebellum were studied between 3 to 12 postnatal days. The connection of these cells with the radial glial fibers of the primitive molecular layer could be established. On this basis it was assumed that the mitotic cells studied were immature Bergmann-glial cells whose proliferative activity seemed to continue even after the formation of their characteristic radial fibers. This phenomenon might offer an explanation for the divergent views on the generation time of Bergmann-glia.Holder of a postgraduate fellowship from the University Medical School of Havana, Cuba.     

Evidence for a GABAergic nigrothalamic pathway in the rat

Summary Unilateral stereotaxic microinjection of muscimol into the caudal region of the substantia nigra (SN) evoked tight, dose-related contralateral locomotor asymmetry and stereotypy. These behaviours were partially attenuated by various pre-treatments, including 6-hydroxydopamine lesions of the nigrostriatal dopamine pathway, intraperitoneal (i.p.) haloperidol, and inhibition of thalamic GABA-transaminase activity by local intrathalamic injection of ethanolamine-O-sulphate. Electrolytic or kainic acid lesions of the medial thalamic nuclei (MTN) partially reduced the contraversive rotation to intranigral muscimol, and completely abolished the similar behaviour elicited by apomorphine (25 g) injected into the ipsilateral caudate nucleus. Contraversive turning to intranigral muscimol was completely inhibited by kainic acid lesions of the ipsilateral SN, but potentiated by intrathalamic injection of picrotoxin. Muscimol (40 ng-4 g) administered to the MTN complex in one hemisphere stimulated rats to move in ipsilateral circles that were unaffected by haloperidol. The results of these behavioural experiments suggest that the nigrostriatal dopamine pathway, the nigrothalamic projection and possibly other non-dopaminergic SN efferents all play important roles in mediating the influences of the SN on motor and stereotyped behaviours. Disruption of the nigrothalamic pathway following electrical or chemical injury to the SN was accompanied by falls in GABA and its synthesising enzyme in the corresponding MTN. These data, together with the findings of our electrophysiological study presented in the following paper, are consistent with the nigrothalamic system having a GABAergic inhibitory function.This work was partly supported by an M.R.C. programme grant awarded to Prof. D.W. StraughanI.C. Kilpatrick and A. Fletcher are respectively M.R.C. and S.R.C. scholars     

Evidence for a GABAergic nigrothalamic pathway in the rat

Extracellular recordings were made from neurones in the ventromedial and parafasicular nuclei of the rat thalamus, many of which had demonstrable capsular or caudate projections. These cells responded to electrical stimulation of the ipsilateral substantia nigra with a short latency (4 ms) inhibition presumed to be monosynaptic. This inhibitory response was often preceded by a brief period of increased excitability (latency approximately 3 ms) attributed to activation of corticofugal collaterals. Longer latency, presumably oligosynaptic excitations (latency approximately 8 ms) and inhibitions (approximately 18 ms) were also obtained, but were more commonly evoked in non-projection neurones. All units were inhibited by iontophoretically applied GABA, glycine or 5-HT. Short and long latency synaptic and GABA-induced inhibitions were selectively blocked by bicuculline. Strychnine only antagonised glycine, while 5-HT was not affected by either convulsant. Intranigral injection of muscimol greatly elevated the spontaneous discharge rate of thalamic neurones, particularly those with a striatal projection. These data are compatible with nigrothalamic neurones maintaining a tonically active, GABA-mediated inhibition of cells in the ventromedial and parafascicular nuclei of the thalamus. It is speculated that intranigral muscimol indirectly activates these thalamic cells and thereby initiates contraversive circling behaviour by suppressing this inhibitory system.