The Utility of 3D Ultramicroscopy for Evaluating Cellular Therapies After Spinal Cord Injury.

Cell therapies have shown promise for repairing the injured spinal cord in experimental models and are now being evaluated in clinical trials for the treatment of human spinal cord injury (SCI). To date, experimental evaluation of implanted cell survival, migration, and integration within the injured central nervous system (CNS) of animals has been technically demanding, requiring tissue sectioning, staining, imaging, and manual reconstruction of 2-dimensional (2D) specimens in 3 dimensions (3D). Not only are these histological procedures laborious and fraught with processing artifacts during manual 3D reconstruction, but they are time-intensive. Herein we describe the utility of 3D ultramicroscopy for assessment of cell therapies after SCI, a new state-of-the-art imaging modality in which whole brain and spinal cord samples are optically sectioned to allow evaluation of intact, macroscopic specimens with microscopic resolution.

The role of chloride transport in postsynaptic inhibition of hippocampal neurons.

Hippocampal inhibitory postsynaptic potentials are depolarizing in granule cells but hyperpolarizing in CA3 neurons because the reversal potentials and membrane potentials of these cells differ. Here the hippocampal slice preparation was used to investigate the role of chloride transport in these inhibitory responses. In both cell types, increasing the intracellular chloride concentration by injection shifted the reversal potential of these responses in a positive direction, and blocking the outward transport of chloride with furosemide slowed their recovery from the injection. In addition, hyperpolarizing and depolarizing inhibitory responses and the hyperpolarizing and depolarizing responses to the inhibitory neurotransmitter gamma-aminobutyric acid decreased in the presence of furosemide. These effects of furosemide suggest that the internal chloride activity of an individual hippocampal neuron is regulated by two transport processes, one that accumulates chloride and one that extrudes chloride.

Infrared videomicroscopy: a new look at neuronal structure and function.

Brain slices were introduced as a standard preparation for neurophysiological experiments some 20 years ago. A drawback of this preparation compared with cell culture has been the difficulty to visualize individual neurones in standard thick slices. This problem has been overcome by the use of infrared videomicroscopy. Neurones in slices can now be visualized in great detail, and neuronal processes can be patch-clamped under direct visual control. Infrared video-microscopy has also been applied successfully to other fields of neuroscience such as neuronal development and neurotoxicity. A further development of infrared videomicroscopy enables one to visualize the spread of excitation in slices making the technique a tool for the direct investigation of neuronal function.

Intrinsic membrane properties of neostriatal neurons can account for their low level of spontaneous activity.

Electroresponsiveness of neostriatal neurons was studied by intracellular recording in a rat brain slice preparation maintained in standard solution or in solution containing K-channel blockers. In standard solution, the neurons fired repetitively at increasing frequencies with increasing amplitude of direct depolarization. The firing pattern was independent of the membrane potential from which firing was induced. In the presence of tetraethylammonium (20 mM), long-lasting (300-500 ms) plateau potentials could be elicited by the injection of short (5-10 ms) current pulses. Plateau potentials persisted in Na-free solution, in the presence of tetrodotoxin (1-3 microM) and if Ca in the perifusate was replaced by Ba. The plateau was blocked by Cd (500 microM). The plateaux were followed by depolarizing after-potentials. When the plateau potential failed due to fatigue, a small slow depolarization of short duration (10-30 ms) was elicited in Na-free or tetrodotoxin-containing solution, which increased in amplitude with membrane hyperpolarization. This slow depolarization was blocked by Cd, indicating that it was also mediated by Ca. By intrastriatal stimulation in the presence of 4-aminopyridine a long-lasting, voltage-dependent depolarization was triggered from the enhanced postsynaptic potential. In contrast, in the presence of tetraethylammonium, postsynaptic potentials were only slightly increased if they were compared at sizes subthreshold for the plateau potentials. It is concluded that neostriatal neurons, although being characterized as "silent" and "non-bursting", possess slow conductances for inward currents which they share with other mammalian central neurons. However, in contrast, to other central neurons, their Ca-spikes are suppressed by their K-conductances and, in contrast to oscillating neurons, low-threshold Ca-potentials are not prominent.

Long-term depression in the basolateral amygdala of the mouse involves the activation of interneurons.

Long-term depression (LTD) in the basolateral amygdala, following low frequency stimulation (1 Hz/900 pulses) of the lateral amygdala, was studied in an in vitro slice preparation of 2-3 weeks and 2-4 months old mice. Whole-cell patch-clamp recordings of neurons, visualized by means of infrared videomicroscopy, and extracellular field potential recordings were performed. Loading single neurons with the calcium chelator BAPTA (30 mM) did not reduce the excitatory postsynaptic currents following low frequency stimulation. However, buffering presynaptic calcium with BAPTA-AM, and application of the specific Ca2+/calmodulin-stimulated protein kinase II antagonist KN-62 (1-[N,O-bis(5-isoquinoline sulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperizine), blocked low frequency stimulation-induced LTD. The induction of LTD was reduced by the competitive N-methyl-D-aspartate receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (50 microM), and blocked by the metabotropic glutamate receptor antagonist (-)-amino-4-carboxy-methyl-phenylacetic acid (1 mM), and by 5-hydroxytryptamine (5-HT; 30 microM) via the activation of 5-HT(1A) receptors. Also blocking GABA(A) receptor-mediated synaptic transmission with bicuculline (10 microM) or picrotoxin (20 microM) reduced the induction of LTD. Visually and electrophysiologically identified interneurons in slices from 2 weeks old mice, expressed in contrast to adult mice (2-4 months), pronounced LTD. Principal neurons showed only weak LTD after low frequency stimulation.A synopsis of these findings suggests a pivotal role of GABAergic interneurons and serotonergic afferents in the induction of LTD in the basolateral nucleus of the amygdala.

Reduction of excitation by interleukin-1 beta in rat neocortical slices visualized using infrared-darkfield videomicroscopy.

The cytokine interleukin-1 beta (IL-1 beta) is thought to be critically involved in the neuroendocrine and behavioral changes which occur in response to systemic infection. In the present study, we have employed the novel technique of infrared-darkfield videomicroscopy to examine the effect of IL-1 beta on the intrinsic optical signal (IOS), an indicator of the spread of neuronal excitation and synaptic transmission in the mammalian central nervous system. Low doses of IL-1 beta delivered exogenously to rat neocortical slices produced a reduction of the area of the column-like IOS evoked by orthodromic stimulation. The effect of IL-1 beta was reversible on washout and not mimicked by heat-inactivated IL-1 beta. These results suggest a possible modulatory role of IL-1 beta on synaptic transmission in the rat neocortex which is probably mediated through an activation of GABAA receptors.

The spread of excitation in neocortical columns visualized with infrared-darkfield videomicroscopy.

A combination of darkfield techniques and infrared videomicroscopy was used to measure the intrinsic optical signal (IOS) in slices of adult rat neocortex. The IOS, which reflects changes in light transmittance and scattering, provides a means of studying the spread of neuronal excitation and its modulation with high sensitivity and spatial resolution. The column-like IOS elicited by orthodromic stimulation is in accordance with models of neocortical circuitry. Blockade of synaptic transmission by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphovaleric acid (D-APV) reduced the IOS. The GABAA agonist muscimol and the neuroactive steroid 5 alpha-tetrahydrodeoxy-corticosterone (5 alpha-THDOC) decreased the spread of excitation, whereas the GABAA antagonist bicuculline increased it. The present data suggest that the spatial spread of excitation in different neocortical layers is delimited by GABAergic inhibition mediated by the activation of GABAA receptors.

Circuitry of rat barrel cortex investigated by infrared-guided laser stimulation.

Infrared-guided laser stimulation was used to examine the synaptic connectivity of neurons in rat barrel cortex. Layer V pyramidal neurons were visualized by infrared videomicroscopy and their membrane potential was recorded with patch pipettes. Presumptive presynaptic neurons were activated by uncaging glutamate with the light of a uv laser directed onto these neurons superfused with medium containing caged glutamate. Synaptic connections were identified by postsynaptic potentials following laser stimulation. The most frequent synaptic connections were found between layer V pyramidal neurons. The probability of this intralaminar input declined monotonically with the lateral distance between stimulated and recorded neuron. In contrast, input from layer II/III onto lamina V neurons showed a periodic organization. Synaptic connections originating from this lamina clearly reflected the barrel structure, with more input originating from the barrel column side, and less input from the barrel column centre. Thus, a barrel-specific organization seems to be especially pronounced for synaptic input from layer II/III to neurons of layer V.

Direct observation of neurotoxicity in brain slices with infrared videomicroscopy.

We employed the novel technique of infrared videomicroscopy to study the morphological changes induced by the neurotoxicity of high concentrations of L-glutamate and by anoxia. The infrared videomicroscopy system described uses an inverted microscope and employs a combination of infrared illumination, differential interference contrast (DIC) and contrast enhancement by video. With this system, we were able to observe swelling of neurons 50 microns deep in rat neocortical slices after bath application of glutamatergic agonists or during anoxia. By recording in time lapse mode it was possible to visualize the dynamics of cell swelling and to demonstrate neuroprotection by glutamatergic antagonists. The method may be of use in screening of potential neuroprotective drugs for stroke therapy.

Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy.

With a combination of infrared illumination, differential interference contrast (DIC) and image intensification by video, unstained living neurons have been visualized up to a depth of 50-100 microns in 300-microns thick brain slices. Recording and application pipettes could be placed under visual guidance. When applied to slices from rat neocortex and hippocampus, pyramidal and non-pyramidal neurons could be differentiated with medium magnification (20x-40x objective). Local neuronal clusters and bundling of apical dendrites of pyramidal cells were visible. The use of an objective with high numerical aperture (63x, N.A. 1.4) allowed the visualization of structures in the submicron range in neocortex and hippocampus. In combination with electrophysiological recordings, infrared DIC-videomicroscopy should facilitate the search for morphological changes underlying neuronal plasticity and the characterization of neuronal networks.

Late development of intrinsic excitation in the rat neostriatum: an in vitro study.

Functional maturation of intrinsic circuitry in the neostriatum was studied by intracellular recording and intracellular staining with Lucifer yellow in slices obtained from rat pups at postnatal days (P)1-20 and from adult rats. The most striking observation was that intrastriatal stimulation elicited predominantly inhibitory responses in slices obtained from animals of P1-6. In contrast, intrastriatally evoked responses in slices obtained after P10 were predominantly excitatory. The inhibitory postsynaptic potentials (IPSPs) recorded in slices obtained from pups were blocked by bicuculline (50 microM) and exhibited a reversal potential at about -60 mV which shifted in depolarizing direction when intracellular Cl- activity increased. Thus, these IPSPs correspond to IPSPs observed in adult animals. It is concluded that maturation of excitatory synapses is the main change during postnatal development. The changes of postsynaptic potentials were paralleled by the appearance of spines on dendrites around P7 as revealed by intracellular staining. The apparent input resistances and time constants of young neurons were very high and responses to large current injections were often distorted by humps which could not be observed in adult neurons. Only young neurons responded with bursts to synaptic activation in the presence of bicuculline (50 microM). It appears that dendritic conductances have a stronger influence on somatic discharge in the electrotonically compact young neurons than in adult neurons.

Actions of noradrenaline on neocortical neurons in vitro.

The effects of noradrenaline (NA) and alpha- and beta-adrenergic agonists on neocortical neurons in rat brain slices were studied by intracellular recording. NA added to the perfusion medium decreased the orthodromically evoked excitatory postsynaptic potential (EPSP) and increased the neuronal excitability on current injection. The decrease of the EPSP by NA was mimicked by alpha-adrenoceptor activation. The beta-adrenergic agonist, isoprenaline, increased the neuronal excitability to current injections similarly to NA. In addition, isoprenaline markedly increased the amplitude of a late component of the EPSP (I-EPSP). The increase of the I-EPSP was long-lasting (up to 90 min). Since this late component of the EPSP is blocked by the NMDA (N-methyl-D-aspartate)-antagonist APV (D-2-amino-5-phosphonovalerate), an enhancement of this excitatory synaptic transient via beta-adrenoceptors may be relevant for the facilitatory effect of NA on neuronal plasticity.

Persisting selective block of unmyelinated fibers in cutaneous nerves of the cat by distilled water.

Experiments were done in vivo on the sural nerve of the cat's hindlimb. The nerve was desheathed for a length of 8 mm and superfused by Tyrode solution or distilled water. Electrical stimulation and recording was used to evaluate conduction block of nerve fibers. Irrigation with distilled water for periods of 3-5 min caused a persisting selective block of conduction in C-fibers while most of the A-fibers were unaffected. Recovery of the C-fibers never occurred during the time of observation (up to 130 min).

Phenol solutions differentially block conduction in cutaneous nerve fibers of the cat.

The action of phenol in Tyrode solution on nerve conduction has been investigated in vivo in the desheathed sural nerve of cats. A section of 8 mm of the nerve was exposed to phenol solution. At low phenol concentrations (0.05-0.125%) a reversible block of A- and C-fibers occurred. At higher concentrations (0.6-1%) a selective and persistent C-fiber block could be achieved. The size of the residual A-volley was between 50% and 81% when all C-fibers were blocked.

Actions of vasoactive intestinal polypeptide (VIP) on neocortical neurons of the rat in vitro.

The action of vasoactive intestinal polypeptide (VIP) was studied using intracellular recording techniques in neocortical neurons of the rat in vitro. When added to the perfusion medium, VIP enhanced direct excitability and increased the amplitude of the excitatory postsynaptic potential (EPSP). Effects of VIP were long-lasting and in most cases not fully reversible within the observation period. The present results suggest a role for VIP in long-lasting peptidergic modulation of excitatory synaptic components as well as direct excitability of neocortical pyramidal neurons of the adult rat.

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation.

Changes in intrinsic optical signal of rat neocortical slices following afferent stimulation were recorded using darkfield infrared-videomicroscopy. Response amplitude was linearly related to stimulation intensity. The intensity of the optical signal reached its maximum 3 s after onset of stimulation and redecayed with a mean time constant of 23 +/- 7.1 s. The optical signal had a columnar shape. The size of the column was independent from stimulation intensity with stimuli of medium amplitudes. The extent of the optical signal corresponded to the extent of the electrical activation. Changes in intrinsic optical properties may be a useful tool for the study of spread of excitation in neuronal tissue in vitro.

Ultramicroscopy: 3D reconstruction of large microscopical specimens.

Ultramicroscopy is a microscopical technique that allows optical sectioning and 3D reconstruction of biological and medical specimens. While in confocal microscopy specimen size is limited to several hundred micrometers at best, using ultramicroscopy even centimeter sized objects like whole mouse embryos can be reconstructed with micrometer resolution. This is possible by using a combination of a clearing procedure and the principle of lightsheet illumination. We present ultramicroscopic 3D reconstructions of whole immunohistochemically labelled mouse embryos and adult Drosophila, giving detailed insight into their anatomy. Its speed and simplicity makes ultramicroscopy ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

[Endogenous cannabinoid system. Effect on neuronal plasticity and pain memory]. / Endogenes Cannabinoidsystem. Einfluss auf neuronale Plastizität und Schmerzgedächtnis.

AIM: The aim of this study was to evaluate the role of the endogenous cannabinoid system in controlling neuroplasticity. METHODS: The pain threshold for electrical stimuli was determined in transgenic mice lacking the cannabinoid receptor type 1 (CB1(-/-)) and in the corresponding respective wild-type animals. Electrophysiological experiments were performed in prepared brain slices to test the effect of endogenous and exogenous cannabinoids on synaptic transmission and long-term potentiation (LTP) in the amygdala. RESULTS: The pain threshold was nearly identical in both groups for the first pain induction; however, with repeated pain induction it decreased to a significantly greater extent in the CB1(-/-) mice than in the wild-type animals. Synoptic transmission and the inducibility of LTP were not influenced by the acute pharmacological blockade of CB1 receptors, but inhibited by the CB1 agonist WIN55,212-2. CONCLUSION: The endogenous cannabinoid system is involved in the control of neuroplasticity as part of pain processing . Cannabinoids prevent the formation of LTP in the amygdala via activation of CB1 receptors. Synoptic transmission and the inducibility of LTP were not influenced by the acute pharmacological blockade of CB1 receptors, but inhibited by the CB1 agonist Win55,212-2.

Muscarinic slow excitation and muscarinic inhibition of synaptic transmission in the rat neostriatum.

Intracellular recording from neurones in rat neostriatal slices was used to compare the muscarinic effects of endogenous acetylcholine (ACh) released from cholinergic neostriatal synapses with the action of exogenously applied muscarinic agonists. Repetitive electrical stimulation in the neostriatum evoked a series of fast excitatory post-synaptic potentials (e.p.s.p.s) followed by a short, variable period of input resistance decrease. In the presence of the acetylcholinesterase (AChE) inhibitor, physostigmine, these potentials were followed by a slow e.p.s.p. which lasted about 60 s. Higher stimulus intensities were needed to elicit the slow e.p.s.p. than the fast e.p.s.p. The slow e.p.s.p. could not be observed after a single stimulus. Its amplitude was graded and increased with stimulus strength. The slow e.p.s.p. was blocked by the muscarinic antagonist atropine (10 microM) and by Ba2+ (100 microM). Input resistance increased during the slow e.p.s.p. Depolarization of the cell increased the size of the slow e.p.s.p. and hyperpolarization decreased it. Simultaneously, resting input resistance increased with membrane depolarization and decreased with membrane hyperpolarization. Repetitive intrastriatal stimulation was followed by a hyperpolarization instead of the depolarization at membrane potentials negative to -75 mV. Input resistance increased during this hyperpolarization as it did during the slow e.p.s.p. The slow e.p.s.p. persisted at membrane potentials of -70 to -80 mV if K+ concentration in the saline was reduced from 5 to 2 mM. In 10 mM-K+, the repetitive stimulation was followed by a hyperpolarization even at membrane potentials as low as -60 to -50 mV. Bath perfusion of high concentrations (100 microM) of muscarine or carbachol induced a sustained increase in the input resistance. The muscarinic agonists also reduced the amplitude of intrastriatally evoked fast e.p.s.p.s; however, this effect was transient and compensated by the increase in input resistance. The effects of the muscarinic agonists on input resistance and e.p.s.p. amplitude were antagonized by atropine (10 microM). Sustained decreases of e.p.s.p. amplitude were induced by the bath application of low doses (0.5-10 microM) of muscarine or carbachol. Input resistance was not altered. Atropine (1-10 microM) antagonized this effect. A sustained reduction of fast e.p.s.p. amplitude resulted also from inhibition of AChE by application of physostigmine (1-100 microM). Input resistance and neuronal excitability were not affected by AChE blockade.(ABSTRACT TRUNCATED AT 400 WORDS)