Post-processing strategies in image scanning microscopy.

Image scanning microscopy (ISM) coupled with pixel reassignment offers a resolution improvement of √2 over standard widefield imaging. By scanning point-wise across the specimen and capturing an image of the fluorescent signal generated at each scan position, additional information about specimen structure is recorded and the highest accessible spatial frequency is doubled. Pixel reassignment can be achieved optically in real time or computationally a posteriori and is frequently combined with the use of a physical or digital pinhole to reject out of focus light. Here, we simulate an ISM dataset using a test image and apply standard and non-standard processing methods to address problems typically encountered in computational pixel reassignment and pinholing. We demonstrate that the predicted improvement in resolution is achieved by applying standard pixel reassignment to a simulated dataset and explore the effect of realistic displacements between the reference and true excitation positions. By identifying the position of the detected fluorescence maximum using localisation software and centring the digital pinhole on this co-ordinate before scaling around translated excitation positions, we can recover signal that would otherwise be degraded by the use of a pinhole aligned to an inaccurate excitation reference. This strategy is demonstrated using experimental data from a multiphoton ISM instrument. Finally we investigate the effect that imaging through tissue has on the positions of excitation foci at depth and observe a global scaling with respect to the applied reference grid. Using simulated and experimental data we explore the impact of a globally scaled reference on the ISM image and, by pinholing around the detected maxima, recover the signal across the whole field of view.

Voltage-dependent calcium signaling in rat cerebellar unipolar brush cells.

Unipolar brush cells (UBCs) are a class of excitatory interneuron found in the granule cell layer of the vestibulocerebellum. Mossy fibers form excitatory inputs on to the paint brush shaped dendrioles in the form of giant, glutamatergic synapses, activation of which results in prolonged bursts of action potentials in the postsynaptic UBC. The axons of UBCs themselves form mossy fiber contacts with other UBCs and granule cells, forming an excitatory, intrinsic cerebellar network that has the capacity to synchronize and amplify mossy fiber inputs to potentially large populations of granule cells. In this paper, we demonstrate that UBCs in rat cerebellar slices express low voltage activated (LVA) fast-inactivating and high voltage activated (HVA) slowly inactivating calcium channels. LVA calcium currents are mediated by T-type calcium channels and they are associated with calcium increases in the dendrites and to a lesser extent the cell soma. HVA currents, mediated by L-type calcium channels, are slowly inactivating and they produce larger overall increases in intracellular calcium but with a similar distribution pattern. We review these observations alongside several recent papers that examine how intrinsic membrane properties influence UBCs firing patterns and we discuss how UBC signaling may affect downstream cerebellar processing.

Nitric oxide release is induced by dopamine during illumination of the carp retina: serial neurochemical control of light adaptation.

Several lines of indirect evidence have suggested that nitric oxide may play an important role during light adaptation of the vertebrate retina. We aimed to verify directly the effect of light on nitric oxide release in the isolated carp retina and to investigate the relationship between nitric oxide and dopamine, an established neuromodulator of retinal light adaptation. Using a biochemical nitric oxide assay, we found that steady or flicker light stimulation enhanced retinal nitric oxide production from a basal level. The metabotropic glutamate receptor agonist L-amino-4-phosphonobutyric acid, inhibited the light adaptation-induced nitric oxide production suggesting that the underlying cellular pathway involved centre-depolarizing bipolar cell activity. Application of exogenous dopamine to retinas in the dark significantly enhanced the basal production of nitric oxide and importantly, inhibition of endogenous dopaminergic activity completely suppressed the light-evoked nitric oxide release. The effect of dopamine was mediated through the D1 receptor subtype. Imaging of the nitric oxide-sensitive fluorescent indicator 4,5-diaminofluorescein di-acetate in retinal slices revealed that activation of D1 receptors resulted in nitric oxide production from two main spatial sources corresponding to the photoreceptor inner segment region and the inner nuclear layer. The results taken together would suggest that during the progression of retinal light adaptation there is a switch from dopaminergic to nitrergic control, probably to induce further neuromodulatory effects at higher levels of illumination and to enable more efficient spreading of the light adaptive signal.

Nitric oxide is required for the induction and heterosynaptic spread of long-term potentiation in rat cerebellar slices.

1. In the cerebellar cortex, brief, 8 Hz activation of parallel fibres (PFs) induces a cyclic adenosine 3'5'-monophosphate (cAMP) and protein kinase A (PKA)-dependent form of long-term potentiation between PFs and Purkinje cells. 2. With 10 mM BAPTA in the recording pipette, potentiation evoked by raised frequency stimulation (RFS) to one of two, synaptically independent PF inputs to the same Purkinje cell did not remain input specific but consistently spread to synapses that did not receive RFS, up to the maximum distance tested of 168 microm. 3. LTP at activated and non-activated sites was accompanied by a decrease in paired pulse facilitation (PPF). The PKA inhibitor H-89 blocked both of these effects. Inhibition of nitric oxide synthase (NOS), either by 7-nitro-indazole (7-NI) or N (G)-nitro-L-arginine methyl ester (L-NAME), completely prevented heterosynaptic potentiation and associated reduction in PPF. LTP at distant synapses was selectively prevented by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). Inhibition of soluble guanylate cyclase or protein kinase G had no effect on either pathway. 4. Synaptic potentiation at PF-PC synapses, induced by the adenylate cyclase activator forskolin, was also prevented by inhibition of NOS. Forskolin-induced increases in mEPSC frequency were similarly prevented by NOS inhibition and mimicked by the NO donor spermine NONOate. 5. These results are consistent with the notion that heterosynaptic potentiation is of pre-synaptic origin and dependent upon activation of cAMP/PKA and NO. Moreover, they suggest that cAMP/PKA activation stimulates NO production and this diffusible messenger facilitates pre-synaptic transmitter release at synapses within a radius of upwards of 150 microm, through a mechanism that does not involve cGMP.

Roles for nitric oxide and arachidonic acid in the induction of heterosynaptic cerebellar LTD.

In cerebellar slices conjunctive pairing of parallel fibre (PF) stimulation with depolarization of Purkinje cells (PCs) induces a long-term depression (LTD) of PF synaptic transmission that spreads to unpaired PF inputs to the same cell. Inhibitors of NO synthase (7-nitro-indazole), soluble guanylate cyclase (ODQ) and PKG (KT5823) all prevented depression at each of two independent PF pathways to a single PC. Inhibition of NOS also unmasked a platelet activating factor (PAF)-mediated synaptic potentiation of possible presynaptic origin. LTD was also prevented by the phospholipase A2 inhibitor OBAA but was rescued by co-perfusion with arachidonic acid. We conclude that NO and diffusible products of phospholipase A2 metabolism are potential mediators of the spread of cerebellar plasticity at the single cell level.

Intercellular action of nitric oxide increases cGMP in cerebellar Purkinje cells.

cGMP is thought to play a role in cerebellar signalling yet its production within Purkinje cells has never been detected. In the present study, the hydrolysis of a fluorescent substrate analogue, 2'-O-anthranyloyl cyclic GMP, by type 5 phosphodiesterase was monitored within Purkinje cells in slices and in culture. Nitric oxide, either endogenously released from adjacent neurons or pharmacologically applied, accelerated the rate of hydrolysis in a manner that was dependent on soluble guanylyl cyclase, demonstrating that nitric oxide triggers cyclic GMP production in Purkinje cells, which in turn activates type 5 phosphodiesterase. We conclude that NO acts as an intercellular messenger in the cerebellar cortex and that parallel fibre terminals are a probable source of nitric oxide.

Receptors, second messengers and protein kinases required for heterosynaptic cerebellar long-term depression.

Raising the frequency and intensity of stimulation to one of two sets of parallel fibre synaptic inputs to cerebellar Purkinje cells results in a localised calcium influx and a long-term depression (LTD) of parallel fibre-Purkinje cell responses. Although the calcium influx remains spatially constrained, depression spreads heterosynaptically to distant sites. Inhibition of the synthetic enzyme for cGMP, guanylate cyclase, did not significantly affect the overall level of calcium-dependent synaptic depression observed at the site of raised stimulation (test site), but it entirely prevented synaptic depression at the distant (control) site. Inhibition of protein kinase G produced identical results. In contrast, protein kinase A inhibition had no effect. Selective inhibition of either metabotropic glutamate receptors (mGluRs), protein kinase C (PKC) or tyrosine protein kinase (PTK) blocked depression at both sites equally effectively. These data reveal that two, inter-dependent cellular pathways capable of inducing cerebellar LTD exist. The levels of PF stimulation required to induce heterosynaptic depression were similar to those used routinely in more widely accepted models of LTD. The data predict that cerebellar long-term depression will not be input specific at the single cell level under those conditions of PF-activation that give rise to NO/cGMP production.

An evaluation of the synapse specificity of long-term depression induced in rat cerebellar slices.

Whole-cell excitatory postsynaptic currents (EPSCs) were recorded from single Purkinje cells (PCs) in rat cerebellar slices in response to alternate activation of two separate sets of parallel fibres (PF1 and PF2). Pairing the stimulation of one input (PF1) with PC depolarisation at 1 Hz for 5 min produced varied effects, including a long-term depression (LTD) of subsequent responses, a medium-term potentiation, or no change relative to baseline levels (n = 14). In all but two cases PF2 responses mirrored those in PF1, in both direction and magnitude even though this second pathway was not specifically activated during pairing. Increasing the stimulus strength to evoke larger amplitude EPSCs (> 1000 pA) dramatically increased the proportion of cells that underwent LTD in both PF1 and PF2. LTD in both pathways was postsynaptic calcium dependent. PC depolarisation alone (n = 7) or PF1 stimulation paired with PC hyperpolarisation (n = 6) failed to induce LTD at either site. Pairing PF1 stimulation with climbing fibre (CF) activation at 1 Hz for 5 min produced LTD in the majority of cells regardless of the strength of PF stimulation. LTD under these conditions was not, however, input specific, even at the lowest stimulus strengths. With EPSCs greater than 1000 pA in amplitude, depression was apparent in both pathways even when the duration of PF1 pairing with depolarisation was limited to 1 min. Full expression of LTD in PF2 required stimulation of this pathway to be resumed within a distinct temporal window of conjunctive pairing with PF1. Introducing a delay of 20 min before resumption of PF2 activation preserved the input specificity of synaptic depression. We conclude that pairing either PC depolarisation or CF activation with stimulation of a discrete set of PFs produces LTD that spreads to adjacent synapses on the same PC.

Peptide fragments of beta-amyloid precursor protein: effects on parallel fiber-Purkinje cell synaptic transmission in rat cerebellum.

The effects of peptide fragments of the beta-amyloid precursor protein (betaAPP) on parallel fiber (PF)-Purkinje cell synaptic transmission in the rat cerebellum were examined. Transient inward currents associated with calcium influx were induced by localized applications of the 105-amino acid carboxy-terminal fragment (CT105) of betaAPP to discrete dendritic regions of intact Purkinje cells. betaAPP and the amyloid beta (Abeta) peptide fragments Abeta1-16, Abeta25-35, and Abeta1-42 had little or no effect. Inward currents were also observed following applications of CT105 to isolated patches of somatic Purkinje cell membrane. All five proteins/peptides induced some depression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor-mediated synaptic transmission between PFs and Purkinje cells, through a combination of pre- and postsynaptic effects. CT105 induced the greatest depression, which spread to distant synapses following local application and which was prevented by inhibition of nitric oxide synthase. These data indicate that CT fragments of the betaAPP can modulate AMPA-mediated glutamatergic synaptic transmission in the cerebellar cortex. These fragments may therefore be considered alternative candidates for some of the neurotoxic effects of Alzheimer's disease.

Light dose and time dependency of photodynamic cell membrane damage.

We have investigated the light dose and time dependency of photodynamic cell membrane damage using electrophysiological methods. This study controls the level of cell membrane damage by precisely administration of the light dose. The photosensitizer used was 5',5"-bis(aminomethyl)-2,2':5',2"-terthiophene dihydrochloride (BAT). A confocal laser scanning microscope was used to provide rapid light activation (< 1 s) and the subsequent membrane damage was monitored using standard patch clamp techniques. In the presence of 49 microM BAT, light levels less than 0.94 J/cm2 led to a reversible depolarization (approximately 20 mV) and reduction of resistance (approximately 10%) within 3 s of illumination. Higher intensities of illumination (> 1.57 J/cm2) caused a complete and irreversible loss of membrane potential and cell membrane resistance within 8 s illumination. The threshold dose of light required to induce cell death by illumination in the presence of BAT was increased in the presence of the antioxidant Trolox-C.

Inhibition of cGMP breakdown promotes the induction of cerebellar long-term depression.

The effects of the nonspecific cyclic nucleotide inhibitors 1-methyl-3-isobutylxanthine (IBMX) and dipyridamole, and the cGMP-specific phosphodiesterase inhibitor Zaprinast were studied on parallel fiber-Purkinje cell synaptic responses in rat cerebellar slices. Bath application of all three compounds, at concentrations shown to inhibit cGMP breakdown, led to stable and robust long-term depression of PF responses. Injections of dipyridamole directly into the Purkinje cell dendrites were similarly effective as bath applications, confirming a postsynaptic site of action. Inhibitors of both protein kinase G and C and also the metabotropic glutamate receptor antagonist MCPG completely prevented the induction of LTD by dipyridamole and Zaprinast. The extent of phosphodiesterase-induced synaptic depression was dependent on the frequency of parallel fiber stimulation, and this form of LTD both occluded and was occluded by LTD induced by pairing parallel and climbing fiber inputs. The degree of LTD induced by IBMX was dose-dependent, and also required PKC and PKG activity, but was preceded by a large, transient potentiation of parallel fiber responses occurring by a postsynaptic mechanism independent of cGMP. These data not only confirm that cGMP is capable of inducing cerebellar LTD when paired with parallel fiber stimulation but indicate that cGMP is an endogenous intermediate in this form of synaptic plasticity.

Strong activation of parallel fibers produces localized calcium transients and a form of LTD that spreads to distant synapses.

The temporal and spatial changes in intracellular calcium levels during separate activation of parallel fiber (PF) and climbing fiber (CF) inputs to cerebellar Purkinje cells were studied. PF stimulation (1 Hz), at relatively high stimulus strengths, led to accumulations of calcium that were similar in peak levels to those following CF stimulation but that remained spatially localized. Such stimuli consistently induced a durable depression of PF synaptic transmission that partially occluded further depression by conventional conjunctive stimuli and that was independent of nitric oxide. This depression was accompanied by a reduction of synaptic efficacy in spatially isolated PF inputs to the same cell that was independent of postsynaptic calcium but that was mediated by nitric oxide. These data indicate that LTD comprises at least two separate processes and that parameters of PF stimulation that are capable of raising calcium levels in Purkinje cell dendrites are also able to induce long-term changes in synaptic efficacy.

NMDA-receptor contribution to spinal nociceptive reflexes: influence of stimulus parameters and of preparatory surgery.

The contribution of NMDA receptors to nociceptive reflexes has been assessed both in awake rats and in electrophysiological tests on alpha-chloralose anaesthetized spinalized rats prepared with different degrees of surgery. Single motor unit activity was recorded in response to alternating noxious mechanical and electrical stimuli applied to one hindpaw, and the results compared with paw pressure withdrawal reflexes in awake rats. There was little contribution by NMDA receptors to nociceptive paw pinch responses either in awake rats or in rats prepared with minimal surgery, but following extensive lumbar surgery the contribution increased significantly to a level similar to that seen in the wind-up component of responses elicited by electrical stimulation. Surgery therefore has effects several segments from the sensory input that it generates. It enhances the NMDA receptor contribution in responses to some but not all types of afferent input.

Step phase-related excitability changes in spino-olivocerebellar paths to the c1 and c3 zones in cat cerebellum.

1. Chronically implanted microwires were used to record extracellular field potentials generated in the c1 and c3 zones in the cortex of lobules V and VI of the cerebellum by non-noxious stimuli delivered to the superficial radial nerve in the ipsilateral forelimb. Responses due to input via climbing fibre afferents were studied; their latency and other characteristics identified them as mediated mainly via the dorsal funiculus spino-olivocerebellar path (DF-SOCP). 2. Responses at individual sites were studied repeatedly with a range of stimulus intensities and during two different behaviours: quiet rest and steady walking on an exercise belt. For responses during walking, step histograms were constructed showing response mean size during different tenths of the step cycle in the ipsilateral forelimb, both in absolute terms and relative to mean size during rest. 3. Step histograms for the same site on different days or different stimulus intensities varied appreciably in form but in both cases the timing of the largest response was usually the same or shifted by only one step tenth. 4. In both zones the largest responses during walking occurred overwhelmingly during the E1 step phase when the limb is extended forwards and down to establish footfall. Least responses were much less uniform in timing but were mostly during stance, particularly its early (E2) part. 5. In many histograms the smallest responses were smaller in mean size than the responses during rest while the largest were larger. These changes were not paralleled by changes in nerve volley size, so presumably reflect step-related central changes in pathway excitability. Facilitations and depressions were differently affected by stimulus intensity and sometimes occurred independently, suggesting generation by separate mechanisms. 6. In both zones there were differences between recording sites which suggests that different DF-SOCP subcomponents innervate different parts of the zones. However, no systematic differences could be firmly established between the medial and lateral subzones of the c1 zone. 7. The results are discussed in relation to the hypothesis that the DF-SOCP constitutes the afferent limb of a transcerebellar mechanism involved in adapting the evolving step.

Induction of cerebellar long-term depression requires activation of glutamate metabotropic receptors.

In rat cerebellar slices, 500 microM (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) reversibly inhibited both dendritic and somatic increases in FLUO-3 fluorescence intensity induced by bath applications of 50-100 microM (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD). No effect of MCPG was observed on dendritically recorded excitatory postsynaptic potentials evoked by synaptic activation of either parallel or climbing fibres. Long-term depression of parallel fibre-Purkinje cell transmission, induced either by conjunctive activation of parallel and climbing fibres or by pairing parallel fibre stimulation with intradendritic injections of 8-BrcGMP, was not only prevented in the presence of MCPG but a robust long-term potentiation of responses consistently occurred. These data show that metabotropic glutamate receptor activation is necessary for the induction of LTD.

cGMP acts within cerebellar Purkinje cells to produce long term depression via mechanisms involving PKC and PKG.

Long term depression (LTD) of parallel fibre-Purkinje cell responses may result from desensitization of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) type glutamate receptors, for which a cascade of reactions involving calcium, inositol-1,4,5-trisphosphate, nitric oxide (NO), guanosine-3'5'-monophosphate (cGMP) and protein kinases C and G has been previously proposed. The involvement of cGMP in synaptic LTD was confirmed by direct injection of cGMP and 8-bromo-cGMP (8-Br-cGMP) into the dendrites of Purkinje cells. Pairing injections with parallel fibre stimulation led to a LTD of synaptic transmission which both occluded and was occluded by heterosynaptically induced LTD. Inhibition of either protein kinases C or G prevented induction of both forms of LTD.

The effect of naloxone on spinal reflexes to electrical and mechanical stimuli in the anaesthetized, spinalized rat.

1. Previous studies of the effects of naloxone on spinal neural responses have yielded disparate results. The reasons for this remain unclear but may relate to the diversity of animal preparations used, the route of administration of naloxone, the site and modality of the stimuli and the intensity of afferent input used. 2. A model requiring little preparative surgery compared to most other electrophysiological preparations has now been used to investigate the effects of naloxone (1, 10, 20 and 50 micrograms kg-1 I.V.) on single-motor-unit flexion reflex responses to alternating mechanical and electrical stimuli in spinalized rats, anaesthetized with alpha-chloralose. 3. Naloxone caused a dose-dependent facilitation of reflex responses to electrical stimuli delivered at intensities sufficient to activate either A fibres alone or A and C fibre afferents together. The component of the responses presumed to be due primarily to activation of C fibres was enhanced relatively more than the A fibre component. 4. Responses evoked during high-intensity mechanical pinch stimuli were not facilitated by equivalent doses of naloxone. The post-stimulus after-discharge was, however, enhanced by a similar percentage to the response to high-intensity electrical stimuli. 5. Lowering the intensity of the mechanical stimulus led to a diminished firing rate of the units during the stimulus itself. The stimulus was, nevertheless, still noxious. Naloxone was found to have a facilitatory effect on this smaller evoked response both during the pinch stimulus and during the period of after-discharge. The apparent lack of effect of naloxone during the higher intensity mechanical stimulus may be due to neurones in the polysynaptic pathway being activated at near-maximal firing rates. 6. We conclude that the ability of naloxone to facilitate spinal reflexes is not dependent on the nature of the stimulus, at least between electrical and mechanical stimuli, but is more a function of the intensity of the applied stimulus.

Spinal effects of four injectable anaesthetics on nociceptive reflexes in rats: a comparison of electrophysiological and behavioural measurements.

1. To assess the direct spinal contributions to the depression of reflexes caused by general anaesthetics, the intravenous potency of four injectable anaesthetics has been compared in two preparations: in decerebrate, spinalised rats, using a novel preparation requiring little surgical intervention, and in intact rats with chronically implanted i.v. cannulae. 2. Methohexitone (1-8 mg kg-1 i.v.), alphaxalone/alphadolone (0.5-8 mg kg-1 i.v.), alpha-chloralose (20-80 mg kg-1 i.v.) and ketamine (0.5-16 mg kg-1 i.v.) all produced a dose-dependent depression of single motor unit activity evoked by controlled noxious mechanical stimuli in decerebrate, spinalised animals. 3. The sedative and motor effects brought about by equivalent doses to those used in the electrophysiological experiments were assessed in intact rats. Methohexitone, alphaxalone/alphadolone and alpha-chloralose all caused similar levels of behavioural sedation at the doses that caused depression of spinal reflexes. Ketamine required relatively much higher doses to cause sedation. 4. To determine whether background anaesthesia modulated the potency with which these compounds affected spinal reflex activity, depressant effects in decerebrate, unanaesthetized rats were compared with those in animals maintained under anaesthesia with either alpha-chloralose or the steroid mixture of alphaxalone/alphadolone. The presence of either of these two agents as maintenance anaesthetics did not influence the effectiveness with which other compounds depressed nociceptive responses. However, additional doses of the maintenance anaesthetics were less effective than the same doses tested in decerebrate animals. 5. All the anaesthetics tested produced a significant depression of spinal reflex responses to noxious stimuli at doses well below those required for anaesthesia. Whilst the presence of maintenance anaesthetics appears not to distort pharmacological tests of other agents, there may nonetheless be a biasing of the samples of cells recorded.