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.

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.

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.

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.

Glutamate receptors form hot spots on apical dendrites of neocortical pyramidal neurons.

Apical dendrites of layer V cortical pyramidal neurons are a major target for glutamatergic synaptic inputs from cortical and subcortical brain regions. Because innervation from these regions is somewhat laminar along the dendrites, knowing the distribution of glutamate receptors on the apical dendrites is of prime importance for understanding the function of neural circuits in the neocortex. To examine this issue, we used infrared-guided laser stimulation combined with whole cell recordings to quantify the spatial distribution of glutamate receptors along the apical dendrites of layer V pyramidal neurons. Focally applied (<10 microm) flash photolysis of caged glutamate on the soma and along the apical dendrite revealed a highly nonuniform distribution of glutamate responsivity. Up to four membrane areas (extent 22 microm) of enhanced glutamate responsivity (hot spots) were detected on the dendrites with the amplitude and integral of glutamate-evoked responses at hot spots being three times larger than responses evoked at neighboring sites. We found no association of these physiological hot spots with dendritic branch points. It appeared that the larger responses evoked at hot spots resulted from an increase in activation of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors and not a recruitment of voltage-activated sodium or calcium conductances. Stimulation of hot spots did, however, facilitate the triggering of both Na+ spikes and Ca(2+) spikes, suggesting that hot spots may serve as dendritic initiation zones for regenerative spikes.

GABA(A) and GABA(B) receptors on neocortical neurons are differentially distributed.

The distribution of functional neurotransmitter receptors on the surface of neurons is highly relevant for synaptic transmission and signal processing. To map functional GABA(A) and GABA(B) receptors on the somadendritic membrane of rat neocortical layer V pyramidal neurons we used patch-clamp recording in combination with infrared-guided laser stimulation to release gamma-aminobutyric acid (GABA) photolytically. The data strongly suggest that relatively more GABA(A) receptors are located at the apical dendrite and relatively more GABA(B) receptors near the soma. Such a specific distribution of GABA(A) and GABA(B) receptors may serve to compensate for differences in electrotonic voltage attenuation between GABA(A) and GABA(B) receptor-mediated inhibitory postsynaptic potentials (IPSPs).

Microglial motility in the rat facial nucleus following peripheral axotomy.

Microglial motility was studied in living mammalian brain tissue using infrared gradient contrast microscopy in combination with video contrast enhancement and time lapse video recording. The infrared gradient contrast allows the visualization of living cells up to a depth of 60 microm in brain slices, in regions where cell bodies remain largely uninjured by the tissue preparation and are visible in their natural environment. In contrast to other techniques, including confocal microscopy, this procedure does not require any staining or labeling of cell membranes and thus guarantees the investigation of tissue which has not been altered, apart from during preparation. Microglial cells are activated and increase in number in the facial nucleus following peripheral axotomy. Thus we established the preparation of longitudinal rat brainstem slices containing the axotomized facial nucleus as a source of activated microglial cells. During prolonged video time lapse recordings, two different types of microglial cell motility could be observed. Microglial cells which had accumulated at the surface of the slice remained stationary but showed activity of the cell soma, developing pseudopods of different shape and size which undulated and which were used for phagocytosis of cell debris. Microglial phagocytosis of bacteria could be documented for the first time in situ. In contrast, ameboid microglia which did not display pseudopods but showed migratory capacity, could be observed exclusively in the depth of the tissue. Some of these cells maintained a close contact to neurons and appeared to move along their dendrites, a finding that may be relevant to the role of microglia in "synaptic stripping", the displacement of synapses following axotomy. This approach provides a valuable opportunity to investigate the interactions between activated microglial cells and the surrounding cellular and extracellular structures in the absence of staining or labeling, thus opening a wide field for the analysis of the cellular mechanisms involved in numerous pathologies of the CNS.

NMDA and AMPA receptors on neocortical neurons are differentially distributed.

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic activation and signal processing in the neocortex. As a novel approach we have used infra-red videomicroscopy in combination with photostimulation or microiontophoresis in brain slices of rat neocortex to map the distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on pyramidal neurons of layer V. Both modes of application revealed a spatially distinct distribution of glutamate receptor subtypes: the soma and the proximal dendrite of neurons are highly sensitive to NMDA, whereas the more distal parts of the dendrite are more sensitive to AMPA. An implication is that NMDA receptors near the soma might regulate the amplification of synaptic signals resulting from AMPA receptor activation on remote dendritic sites.

Visualization of neuronal form and function in brain slices by infrared videomicroscopy.

As a standard preparation for neurophysiological experiments, brain slices were introduced some 20 years ago. Although this technique has greatly advanced our understanding of brain physiology, the utility of this preparation has been limited to some extent by the difficulty of visualizing individual neurons in standard thick slices. The use of infrared videomicroscopy has solved this problem. It is now possible to visualize neurons in slices in great detail, and neuronal processes can be patch-clamped under visual control. Infrared videomicroscopy has also been applied successfully to other fields of neuroscience, such as neuronal development and neurotoxicity. A further development of infrared videomicroscopy allows the visualization of the spread of excitation in slices, making the technique a tool for investigating neuronal function and the pharmacology of synaptic transmission.

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.

The intrinsic optical signal evoked by chiasm stimulation in the rat suprachiasmatic nuclei exhibits GABAergic day-night variation.

Infrared light transmittance imaging was used in rat hypothalamic slices to record an intrinsic optical signal (IOS) of the cell ensemble in the suprachiasmatic nuclei (SCN), the locus of the endogenous circadian clock. Upon optic chiasm stimulation, a transient IOS was observed in an area conforming to the known retinohypothalamic tract innervation in the ventral SCN. An increase in extracellular Mg2+ concentration to 10 mM reduced th IOS, suggesting that the elicited IOS is dependent on synaptic transmission. D-2-amino-5-phosphonopentanoic acid and muscimol suppressed the elicited IOS, indicating that NMDA and GABAA receptor-mediated mechanisms were involved in cell ensemble activity reflected in the IOS. The extracellularly recorded spiking of SCN neurons located outside the IOS area remained largely unaffected by the afferent stimulus. Neurons located within the IOS area responded with a depressed electrical discharge, manifesting an inverse relationship between single-unit discharge and the optical measure. The influence of the endogenous circadian rhythm on the elicited IOS was assessed by carrying out daytime-dependent concentration-response experiments. NMDA and non-NMDA receptor specific compounds did not exhibit significant day-night differences, whereas GABA-specific ligands showed a significant day-night variation in activity. The competitive GABAA receptor antagonist bicuculline enhanced the IOS exclusively in the daytime SCN. 5alpha-Pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), a neuroactive steroid that potentiates GABAergic inhibition, suppressed the IOS in the night-time SCN more than in the daytime SCN. This suggests that in the rat the level of extracellular GABA is higher in night-time SCN compared to the daytime SCN.

Domain-specific antibodies against the B2 chain of laminin inhibit neuronal migration in the neonatal rat cerebellum.

Although the spatial and temporal patterns of neuronal migration have been analyzed in great detail, little direct evidence is available as to what extracellular matrix molecules are involved. Because there is indirect evidence implicating the extracellular matrix protein laminin in neuronal migration, we investigated the effects of antibodies against a synthetic peptide derived from a neurite outgrowth domain of the B2 chain of laminin on neuronal migration in living cerebellar slices. We show by using infrared video microscopy that divalent Fab2 fragments of these antibodies inhibit granule neuronal movement in living slices of (P8) rat cerebellum. This inhibition of neuronal movement manifests itself by cessation of both radial and horizontal translocations of nuclei inside the granule neuronal processes. Fab2 fragments of antibodies against the intact (native) laminin molecule or Fab2 fragments from the preimmune serum do not affect nuclear translocation. Immunocytochemistry shows binding of the divalent Fab2 fragments of the B2 chain-specific antibodies to the Purkinje and Bergmann glial cell areas, and as punctate deposits in between the cells of the external granule cell layer. Native laminin antibodies bind to the basement membranes, and binding of the Fab2 fragments from the preimmune sera cannot be demonstrated. These results indicate that neuronal migration in the postnatal rat cerebellum in vivo involves nuclear translocation that can be inhibited by antibodies against a neurite outgrowth domain of the B2 chain of laminin. Thus, migration of cerebellar granule neurons may depend on the interaction between a neurite outgrowth domain of the B2 chain of laminin and neuronal cytoskeleton involved in nuclear movement.

Novel forms of neuronal migration in the rat cerebellum.

Infrared video microscopy of neonatal rat cerebellum (P0-P14) was used to directly visualize migrating granule neurons in relation to other cerebellar cells in a brain slice for up to 24 hr. Initially (P0-P5), granule neurons move along radial migration pathways of other neuronal fibers. These pathways are probably established by the bipolar granule neurons that attach to the external basement membrane via one process and extend another process toward the Purkinje cell layer. At P5-P8, a substantial number of granule neurons move horizontally and extend long parallel fibers. Both radially and horizontally migrating granule neurons move by nuclear translocation inside their preformed processes with a speed that varies between 6 and 120 microns/hr. In P10-P12 animals, the horizontally oriented granule neurons start to migrate radially. They move into the internal granule cell layer either along the radial pathways of other neuronal fibers or in contact with the matured glial processes. The radial neuronal migration pathways disappear by P14 whereas the glial cell processes are maintained and reach the basal lamina. These results describe novel radial and horizontal modes of neuronal migration that proceed independently of the physical glial guidance.

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.

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.

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.

Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy.

A description is given of the implementation of infrared differential interference contrast (IR-DIC) video microscopy to an upright compound microscope. Using the improved resolution offered by IR-DIC a procedure is described for making patch-pipette recordings from visually identified neuronal somata and dendrites in brain slices. As an example of the application of this technique to electrophysiological recordings from small neuronal processes in brain slices we describe whole-cell current-clamp and cell-attached and excised patch-clamp recordings from the apical dendrites of layer V pyramidal neurons in slices of rat neocortex.