Activity In Cerebellar Cortex Research Articles

Multimodal patterns of inhibitory activity in cerebellar cortex

In this issue of Neuron, Gurnani and Silver (2021) report that activity across Golgi cells, a major type of inhibitory interneuron in the cerebellar cortex, is multidimensional and modulated by behavior. These results suggest multiple functions for inhibition in cerebellar computations.

N-methyl-D-Aspartate Receptors Contribute to Complex Spike Signaling in Cerebellar Purkinje Cells: An In vivo Study in Mice.

N-methyl-D-aspartate receptors (NMDARs) are post-synaptically expressed at climbing fiber-Purkinje cell (CF-PC) synapses in cerebellar cortex in adult mice and contributed to CF-PC synaptic transmission under in vitro conditions. In this study, we investigated the role of NMDARs at CF-PC synapses during the spontaneous complex spike (CS) activity in cerebellar cortex in urethane-anesthetized mice, by in vivo whole-cell recording technique and pharmacological methods. Under current-clamp conditions, cerebellar surface application of NMDA (50 μM) induced an increase in the CS-evoked pause of simple spike (SS) firing accompanied with a decrease in the SS firing rate. Under voltage-clamp conditions, application of NMDA enhanced the waveform of CS-evoked inward currents, which expressed increases in the area under curve (AUC) and spikelet number of spontaneous CS. NMDA increased the AUC of spontaneous CS in a concentration-dependent manner. The EC50 of NMDA for increasing AUC of spontaneous CS was 33.4 μM. Moreover, NMDA significantly increased the amplitude, half-width and decay time of CS-evoked after-hyperpolarization (AHP) currents. Blockade of NMDARs with D-(-)-2-amino-5-phosphonopentanoic acid (D-APV, 250 μM) decreased the AUC, spikelet number, and amplitude of AHP currents. In addition, the NMDA-induced enhancement of CS activity could not be observed after α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors were blocked. The results indicated that NMDARs of CF-PC synapses contributed to the spontaneous CS activity by enhancing CS-evoked inward currents and AHP currents.

In Vivo Calcium Imaging of Circuit Activity in Cerebellar Cortex

In vivo two-photon calcium imaging provides the opportunity to monitor activity in multiple components of neural circuitry at once. Here we report the use of bulk-loading of fluorescent calcium indicators to record from axons, dendrites, and neuronal cell bodies in cerebellar cortex in vivo. In cerebellar folium crus IIa of anesthetized rats, we imaged the labeled molecular layer and identified all major cellular structures: Purkinje cells, interneurons, parallel fibers, and Bergmann glia. Using extracellular stimuli we evoked calcium transients corresponding to parallel fiber beam activity. This beam activity triggered prolonged calcium transients in interneurons, consistent with in vitro evidence for synaptic activation of N-methyl-d-aspartate receptors via glutamate spillover. We also observed spontaneous calcium transients in Purkinje cell dendrites that were identified as climbing-fiber-evoked calcium spikes by their size, time course, and sensitivity to AMPA receptor antagonist. Two-photon calcium imaging of bulk-loaded cerebellar cortex is thus well suited to optically monitor synaptic processing in the intact cerebellum.

Cerebellum and M1 interaction during early learning of timed motor sequences

We used positron emission tomography (PET) to examine within-day learning of timed motor sequences. The results of this experiment are novel in showing an interaction between cerebellum and primary motor cortex (M1) during learning that appears to be mediated by the dentate nucleus (DN) and in demonstrating that activity in these regions is directly related to performance. Subjects were scanned during learning (LRN) across three blocks of practice and during isochronous (ISO) and perceptual (PER) baseline conditions. CBF was compared across blocks of learning and between the LRN and baseline conditions. Results demonstrated an interaction between the cerebellum and M1 such that earlier, poorer performance was associated with greater activity in the cerebellar hemispheres and later, better performance was associated with greater activity in M1. Inter-regional correlation analyses confirmed that as CBF in the cerebellum decreases, blood flow in M1 increases. Importantly, these analyses also revealed that activity in cerebellar cortex was positively correlated with activity in right DN and that DN activity was negatively correlated with blood flow in M1. Activity in the cerebellar hemispheres early in learning is likely related to error correction mechanisms which optimize movement kinematics resulting in improved performance. Concurrent DN activity may be related to encoding of this information and DN output to M1 may play a role in consolidation processes that lay down motor memories. Increased activity in M1 later in learning may reflect strengthening of synaptic connections associated with changes in motor maps that are characteristic of learning in both animals and humans.

Reliable control of spike rate and spike timing by rapid input transients in cerebellar stellate cells

Granule cell activity in cerebellar cortex directly excites Purkinje cells via parallel fibers, but it also inhibits Purkinje cells via cerebellar cortical interneurons. This contribution of inhibitory interneurons to cerebellar cortical processing remains poorly understood. In the present study we examined the response properties of stellate cells in vitro to input patterns that may result from granule cell activity in vivo. We constructed input waveforms that represented the sum of inputs from all individual synapses and applied these waveforms to the soma of stellate cells during whole cell recordings in acute brain slices. The stimulus waveforms contained fluctuations in a broad range of frequencies and were applied at different amplitudes. To determine the contribution of synaptic shunting to stellate cell spike responses we applied the same input waveforms either as a simulated synaptic conductance using dynamic clamping or as a direct current injection stimulus. Only the dynamic clamp stimulus has the shunting properties of real synapses, i.e. leads to different-sized synaptic current as a function of membrane potential. We found that stellate cells spike with millisecond precision in response to fast temporal fluctuations in the total synaptic input. Transient increases in excitatory input frequency led to pronounced stellate cell spike responses, indicating that this pathway may be very responsive to even small assemblies of co-activated granule cells. This was observed regardless of whether the input waveform was applied as a conductance with dynamic clamping, or as a direct current injection. Thus the shunting properties of a conductance input did not play a major role in determining the control of precisely timed spiking. In contrast, a more tonic increase in excitatory conductance did not lead to a sustained spike response as obtained with prolonged positive current injection. However, even with tonic current injection the precision of spiking was lost, as previously observed. Overall, the synaptic response function of stellate cells suggests that this cell type may pick out transients in granule cell activity, and may generate precisely timed inhibition of Purkinje cells during behavior.

Role of climbing fibers in determining the spatial patterns of activation in the cerebellar cortex to peripheral stimulation: an optical imaging study

The spatial patterns of activation in the rat cerebellar cortex evoked by ipsilateral face stimulation were mapped using optical imaging based on the pH sensitive dye, Neutral Red. The aims of the study were to characterize the optical responses evoked by peripheral stimulation and test the hypothesis that the resultant parasagittal banding is due to climbing fiber activation. In the anesthetized rat Crus I and II of the cerebellar cortex were stained with Neutral Red. Epi-fluorescent changes produced by a train of stimuli (5–10 s and 4–20 Hz) to the ipsilateral face were monitored in time using a fast, high resolution charge-coupled device camera. The patterns of activation were quantified using a two-dimensional fast Fourier transform analysis that removed signals with high spatial frequencies and minimized the contribution of horizontal structural elements (i.e. blood vessels). The dominant spatial pattern of activation evoked by face stimulation was that of parasagittal bands. The bands were highly frequency-dependent and were elicited most strongly by stimulus frequencies in the range of 6–8 Hz. There was a large fall-off in the response for frequencies above and below. The optical signal evoked by face stimulation built up over a period of 10 s and then gradually decayed. Within a folium the individual parasagittal bands exhibited some frequency and temporal specificity. Stimulation of the contralateral inferior olive also resulted in the activation of parasagittal bands with characteristics similar to the bands evoked by face stimulation, including a preferred stimulus frequency which peaked at 10 Hz. Injection of lidocaine into the contralateral inferior olive blocked the parasagittal bands evoked by ipsilateral face stimulation, while control injections of saline had no effect. The results confirm that a parasagittal banding pattern is a dominant feature of the functional architecture of the cerebellar cortex. The parasagittal banding pattern observed with Neutral Red is due primarily to the activation of climbing fiber afferents. The frequency tuning of the responses, with the preference for peripheral stimuli of 6–8 Hz, is in agreement with previous findings that the inferior olive is inherently rhythmic. These observations support the hypothesis that inferior olivary neurons are dynamically coupled into groups that activate parasagittal bands of Purkinje cells in the cerebellar cortex. The frequency tuning also supports the hypothesis that the climbing fiber system is involved with timing. Activation of this afferent system may require stimuli with appropriate frequency content and stimuli synchronized to the rhythmicity of the inferior olive.

Functional recruitment of red blood cells to rat brain microcirculation accompanying increased neuronal activity in cerebellar cortex.

Scanning laser-Doppler flowmetry (SLDF) combines laser-Doppler flowmetry and laser scanning to provide images of cerebral blood flow (CBF) with high spatial and temporal resolution. We investigated the contribution of single vascular elements to the local increase of CBF accompanying increased neuronal activity in halothane-anesthetized rats. CBF was examined in the cerebellar cortex under control conditions and in response to electrical stimulation of parallel and climbing fibers. At rest, arterioles contributed 9%, venules 11-13% and small vessels (< 20 microm) 8-14%, while the background constituted 64-72% of the total SLDF signal. During activation the background signal decreased to 55-60% while the signal from arterioles increased to 11-12%, from venules to 14-15% and from small vessels to 14-19%. The signal increase in small vessels that did not give any laser-Doppler signal at rest was due to functional recruitment of red blood cells to the capillary bed. We conclude that functional recruitment may be an integral part of the hemodynamic response accompanying neuronal activity.

Local and propagated vascular responses evoked by focal synaptic activity in cerebellar cortex.

We investigated the local and remote vascular changes evoked by activation of the cerebellar parallel fibers (PFs). The PFs were stimulated (25-150 microA, 30 Hz) in halothane-anesthetized rats equipped with a cranial window. The changes in arteriolar and venular diameter produced by PF stimulation were measured with the use of a videomicroscopy system. Cerebellar blood flow (BFcrb) was monitored by laser Doppler flowmetry and the field potentials evoked by PF stimulation were recorded with the use of microelectrodes. PF stimulation increased the diameter of local arterioles (+26 +/- 1%, mean +/- SE) in the activated folium (n = 10, P < 0.05). The vasodilation was greatest in smaller arterioles (16.5 +/- 0.8 microm), was graded with the intensity of stimulation, and was less marked than the vasodilation produced by hypercapnia in comparably sized vessels (+58 +/- 5%, CO2 pressure = 50-60 mmHg, n = 8). In addition, the vasodilation was greatest along the horizontal beam of activated PFs and was reduced in arterioles located away from the stimulated site in a rostrocaudal direction. The increases in vascular diameter were associated with increases in BFcrb in the activated area (+55 +/- 4%, n = 5). PF stimulation increased vascular diameter (+10 +/- 0.5%, n = 10) also in larger arterioles (30-40 microm) located in the folium adjacent to that in which the PFs were stimulated. Higher-order branches of these arterioles supplied the activated area. No field potentials were evoked by PF stimulation in the area where these upstream vessels were located. The data suggest that increased synaptic activity in the PF system produces a "local" hemodynamic response mediated by synaptic release of vasoactive agents and a "remote" response that is propagated to upstream arterioles from vessels residing in the activated folium. These propagated vascular responses are important in the coordination of segmental vascular resistance that is required to increase flow effectively during functional brain hyperemia.

Obligatory role of NO in glutamate-dependent hyperemia evoked from cerebellar parallel fibers

Electrical stimulation of cerebellar parallel fibers (PF) increases cerebellar blood flow (BFcrb), a response that is attenuated by glutamate receptor antagonists and NO synthase (NOS) inhibitors. We investigated whether administration of NO donors could counteract attenuation by NOS inhibitors of vasodilation produced by PF stimulation. In halothane-anesthetized rats the cerebellar cortex was exposed and superfused with Ringer solution. PF were stimulated with microelectrodes (100 microA, 30 Hz), and BFcrb was recorded by a laser-Doppler probe. During Ringer superfusion, PF stimulation increased BFcrb by 56 +/- 7% and hypercapnia by 72 +/- 5% (n = 5). Superfusion with the nonselective NOS inhibitor N-nitro-L-arginine (L-NNA, 1 mM) reduced resting BFcrb and attenuated the response to PF stimulation (-47 +/- 5%) and hypercapnia (-46 +/- 7%; PCO2 = 50-60 mmHg). After L-NNA, superfusion with the NO donors 3-morpholinosydnonimine (100 microM, n = 5) or S-nitroso-N-acetyl-penicillamine (5 microM, n = 5) reestablished resting BFcrb (P > 0.05 vs. before L-NNA) and reversed L-NNA-induced attenuation of the response to hypercapnia (P > 0.05 vs. before L-NNA) but not PF stimulation (P > 0.05 vs. after L-NNA). Similar results were obtained when NOS activity was inhibited with the inhibitor of neuronal NOS 7-nitroindazole (50 mg/kg i.p.). Like NO donors, the guanosine 3',5'-cyclic monophosphate analog 8-bromoguanosine 3',5'-cyclic monophosphate (n = 5), administered after L-NNA, restored resting BFcrb and counteracted inhibition of the response to hypercapnia but not PF stimulation. In contrast to NO donors and 8-bromoguanosine 3',5'-cyclic monophosphate, the NO-independent vasodilator papaverine (100 microM, n = 5) had no effect on attenuation of responses to PF stimulation or hypercapnia. Thus NO donors are unable to reverse the effect of NOS inhibition on vasodilation produced by PF stimulation. The data support the hypothesis that the vascular response to PF stimulation, at variance with hypercapnia, requires NOS activation and NO production. Thus NO plays an obligatory role in vasodilation produced by increased functional activity in cerebellar cortex.

Lateral cerebellar hemispheres actively support sensory acquisition and discrimination rather than motor control.

This study examined a new hypothesis proposing that the lateral cerebellum is not activated by motor control per se, as widely assumed, but is engaged during the acquisition and discrimination of tactile sensory information. This proposal derives from neurobiological studies of these regions of the rat cerebellum. Magnetic resonance imaging of the lateral cerebellar output nucleus (dentate) of humans during passive and active sensory tasks confirmed four a priori implications of this hypothesis. Dentate nuclei responded to cutaneous stimuli, even when there were no accompanying overt finger movements. Finger movements not associated with tactile sensory discrimination produced no dentate activation. Sensory discrimination with the fingers induced an increase in dentate activation, with or without finger movements. Finally, dentate activity was greatest when there was the most opportunity to modulate the acquisition of the sensory tactile data: when the discrimination involved the active repositioning of tactile sensory surface of the fingers. Furthermore, activity in cerebellar cortex was strongly correlated with observed dentate activity. This distinct four-way pattern of effects strongly challenges other cerebellar theories. However, contrary to appearances, neither our hypothesis nor findings conflict with behavioral effects of cerebellar damage, neurophysiological data on animals performing motor tasks, or cerebellar contribution to nonmotor, perceptual, and cognitive tasks.

Neural mechanisms of blood flow regulation during synaptic activity in cerebellar cortex.

1. We investigated the neural mechanisms of the increases in blood flow produced by synaptic activity using the parallel fiber (PF) system of the cerebellum as a model. The midline cerebellum was exposed in anesthetized rats and the PFs were stimulated with tungsten microelectrodes. Cerebellar blood flow (BFcrb) was recorded using a laser-Doppler probe, whereas field potentials were recorded using glass micropipettes. PF stimulation produced increases in BFcrb that were related to the frequency and intensity of stimulation (+60 +/- 9%, mean +/- SE, at 100 microA and 30 Hz; n = 6). The greatest increases were confined to a band stretching along the major axis of the stimulated folium and corresponding to the beam of activated PFs. The increase in evoked by PF stimulation was associated with a corresponding increase in glucose utilization, assessed by the 2-deoxyglucose method. The increases in BFcrb and the field potentials evoked by PF stimulation were abolished by tetrodotoxin (1 microM; n = 6). Ringer solution containing 12 mM Mg2+ and 0 mM Ca2+ blocked synaptic activity in the PFs and abolished the increases in flow (P > 0.05 from baseline; n = 5). The broad-spectrum glutamate receptor antagonist kynurenate (5 mM) prevented depolarization of Purkinje cells and interneurons and abolished the increase in BFcrb evoked by PF stimulation (P > 0.05; n = 6). Treatment with tetrodotoxin, Mg2+, or kynurenate did not affect the increase in BFcrb elicited by systemic hypercapnia or by topical application of the nitric oxide donor 3-morpholino sydnonimine (P > 0.05 from Ringer solution). We conclude that the increases in flow produced by synaptic activity are linked to glutamate-induced depolarization of Purkinje cells and interneurons. These findings provide evidence that activation of glutamate receptors participates in the mechanisms of functional hyperemia, and they support the validity of the PF system as a model for study of the relationship between synaptic activity and blood flow in the CNS.

Nitric oxide and adenosine mediate vasodilation during functional activation in cerebellar cortex

Activation of the cerebellar parallel fibers (PF) releases glutamate and leads to depolarization of Purkinje cells and interneurons. These cells, in turn, release GABA. We have studied the role of glutamate, GABA, nitric oxide (NO) and adenosine in the increases in cerebellar cortex blood flow (BF crb) elicited by PF stimulation. In anesthetized rats (halothane 1%) the cerebellar vermis was exposed and the site was superfused with Ringer (37°C, pH 7.4). The PF were stimulated electrically (50–100 μA; 30 Hz) and the increases in BF crb were recorded using a laser-Doppler flowmeter. Field potentials were recorded using glass microelectrodes. During Ringer superfusion, PF stimulation increased BF crb by 58 ± 5% ( P < 0.001; analysis of variance; n = 6). Superfusion with the broad spectrum glutamate receptor antagonist kynurenic acid (Kyn; 5 mM) abolished the negative component of the field potential ( n = 4), a finding reflecting lack of depolarization of Purkinje cells and interneurons, and blocked the increase in BF crb ( P > 0.05 from Ringer; n = 6). In contrast, Kyn did not influence the increase in BF crb evoked by hypercapnia (pCO 2 55.4 ± 1.1 mmHg) or by superfusion with the NO donor SIN-1 (0.1, 1 mM; P > 0.05; n = 6). Superfusion with the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT; 100 μM) reduced the elevation in BF crb by 45 ± 4% ( P < 0.05; n = 6) and co-application of 8-SPT and of the NO synthase inhibitor nitro- l-arginine ( l-NA; 1 mM) attenuated the vasodilation further (−82 ± 4% from Ringer; P < 0.01 from 8-SPT alone). The vasodilation elicited by SIN-1 ( n = 6) and the field potentials evoked by PF stimulation ( n = 4) were not affected by 8-SPT or by 8-SPT plus l-NA. The GABA receptor antagonist bicuculline (300 μM) produced the expected changes in the field potentials ( n = 4) but did not affect the vascular response to PF stimulation ( P . 0.05; n = 5). The data suggest that release of glutamate from PF leads to production of NO and adenosine in Purkinje cells and/or interneurons. NO and adenosine may mediate the vasodilation by diffusing toward resistance arterioles and producing smooth muscle relaxation.

Decreased insulin-like growth factor I-mediated protein tyrosine phosphorylation in human olivopontocerebellar atrophy and lurcher mutant mouse

We examined insulin-like growth factor I (IGF-I)-dependent phosphorylation and protein tyrosine kinase (PTK) activity in cerebellar cortex of normal humans, patients with olivopontocerebellar atrophy (OPCA) (“C” kindred) and in lurcher mutant mouse, a suggested animal model for OPCA. PTK activity and IGF-I-dependent protein tyrosine phosphorylation was significantly reduced in cerebellar cortex of human OPCA patients as compared to the normal controls. Immunoblot analysis also demonstrated a decrease in cerebellar 80 kDa phosphotyrosine protein in these patients. By autoradiography, IGF-I receptors were localized in the molecular layer of 30-day-old control and lurcher mutant mice cerebella. However, the lurcher mutant mice showed a decrease in [ 125I]-IGF-I binding in the molecular layer as compared to the littermate controls. The IGF-I receptor autophosphorylation was also markedly reduced in 15-day- and 22-day-old lurcher cerebella. These results suggest that the process of cerebellar degeneration in human OPCA and lurcher mutant mouse may be associated with altered IGF-I receptor binding and protein tyrosine phosphorylation.

Possible CS and US pathways for rabbit classical eyelid conditioning: Electrophysiological evidence for projections from the pontine nuclei and inferior olive to cerebellar cortex and nuclei

Projections from the lateral region of the pontine nuclei and the dorsal accessory inferior olive to both cerebellar cortex and cerebellar dentate/interpositus nuclei were electrophysiologically examined using single-pulse stimulation and single-unit and population recording. Stimulation of the pontine nuclear region activated population potentials and single units recorded in both cerebellar cortex and deep nuclei. Pontine-evoked activity in cerebellar cortex (Larsell's lobule HVI and adjacent areas) was rather well-defined and strong while pontine-evoked activity in the deep cerebellar nuclei seemed relatively more diffuse and weaker. Short onset latencies for both single units and population potentials were found suggesting direct projections. Similar to previous studies, inferior olive stimulation evoked short-latency responses in cerebellar cortex and nuclei thus suggesting direct projections. More pontine- and olivary-evoked activity was seen in cortex than in the nuclei with slightly more olivary-evoked potentials per recording electrode penetration observed than pontine-evoked activity. Our findings suggest that cortical and nuclear regions of the cerebellum receive converging projections from the pontine nuclei and inferior olive, projections that may carry information about stimuli used during classical conditioning. These findings are discussed in terms of cerebellar circuits that may be involved in classical eyelid conditioning.

Optical imaging of parallel fiber activation in the rat cerebellar cortex: Spatial effects of excitatory amino acids

Using optical imaging, the effects of excitatory amino acids and their antagonists on the spatial pattern of activity evoked by the stimulation of parallel fibers in the cerebellar cortex of the anesthetized rat were examined. A vermal folium was stained with the stryrl voltage-sensitive dye RH-795 and imaged with a cooled charge-coupled service system during the activation of the parallel fibers with surface stimulation. Stimulation of the cerebellar cortex produced discrete “beams” of optical activity consistent with extracellular field recordings. The signal-to-noise ratio was excellent (> 10:1), reducing the number of stimuli, exposure time, and acquisition time needed to produce images. Extracellular field potential recordings were used to assay neuronal activity as well as the effects of the various excitatory amino acid agonists. Application of several glutamate receptor antagonists reversibly blocked the optical signals as well as the synaptic components of the extracellular field potentials. Neither N-methyl- d-aspartate nor its antagonist, (±)-2-amino-7-phosphonoheptanoic acid, had any affect on the optical signals or field potentials. These results indicate that the optical signal is not due to the evoked parallel fiber activity, but is generated mainly by postsynaptic targets and the parallel fiber synaptic action is primarily mediated by non-N-methyl- d-aspartate receptors. Other excitatory amino acid agonists had a differential effect on the optical response. Glutamate and kainate increased the “on beam” optical signal evoked by parallel fiber stimulation, in amplitude and width. In contrast, quisqualate always decreased the amplitude and width of the optical beam. Also, quisqualate produced an increase in fluorescence lateral to the optical beam, possibily due to an increase in “off beam” inhibitory activity. The changes in the extracellular field potentials were in agreement with the effects on the optical signals. Two possible mechanisms are proposed to account for the inhibitory effect of quisqualate. One is that quisqualate desensitizes Purkinje cell receptors, the other is that inhibitory interneurons in the cerebellar cortex are more preferentially excited with quisqualate application which in turn inhibits Purkinje cells both “on beam” and “off beam”. In conclusion, voltage-sensitive dye optical signals evoked by stimulation of the cerebellar surface were imaged at high signal-to-noise levels using a cooled charge-coupled device system. Use of excitatory amino acid agonists and antagonists demonstrated that the optical signal was dependent on postsynaptic activity and confirmed that the parallel fiber postsynaptic action is primarily mediated by non-N-methyl- d-aspartate receptors. The reported differential action of glutamate, quisqualate and kainate indicates how different excitatory amino acid receptor classes are involved in the control of spatial activity in the cerebellar cortex.

Changes in extracellular potassium and calcium in rat cerebellar cortex related to local inhibition of the sodium pump

Extracellular K+, Ca2+, and Na+ ([K+]e, [Ca2+]e, [Na+]e) were recorded with ion selective microelectrodes in the cerebellar cortex of urethane-anesthetized rats. Superfusion of the cerebellum with artificial cerebrospinal fluid containing K-strophanthidin (10(-6)-10(-4) mol/l) or other cardioactive steroids, known to be inhibitors of the sodium/potassium pump, had the following effects: elevation of resting [K+]e, reduction of poststimulus K+-undershoots, decrease of resting [Ca2+]e and [Na+]e. For instance, at 3 X 10(-5) mol/l K-strophanthidin within the superfusion solution (the unknown intracerebellar concentration being certainly much smaller), [K+]e was elevated up to 130% and [Ca2+]e reduced to 70% of their resting values. Iontophoretic K+-pulses were enhanced in amplitude at the same time. Control experiments with iontophoretic TMA application demonstrated that the glycoside effects were not due (or in higher concentrations only partly due) to shrinkage of the extracellular fluid volume. When tetrodotoxin (10(-7) mol/l) or Mn2+ (1-3 mmol/l) were additionally superfused, K-strophanthidin effects were qualitatively similar, though quantitatively smaller. This indicates that part of the effects were indirect via neuronal activity evoked by the blockade of the sodium pump. The experiments show that reduction of sodium pump activity in cerebellar cortex has rapid and serious consequences on the distribution of potassium and calcium in the extracellular space, resulting in an alteration of neuronal circuit excitability.

Improved histological localization of GABA-transaminase activity in rat cerebellar cortex after aldehyde fixation.

A method for the chemical fixation of the enzyme GABA-transaminase in nervous tissue is described. It is shown that after perfusion with a formaldehyde/glutaraldehyde fixative, activity of the enzyme in cerebellar cortex is demonstrable whilst cellular morphology is preserved. Results from the improved technique have shown new sites of GABA-transaminase activity in cerebellar cortex. In view of these results a special function for glial cells in this area of brain has been suggested.