Making waves: initiation and propagation of corticothalamic Ca2+ waves in vivo.

Corticothalamic slow oscillations of neuronal activity determine internal brain states. At least in the cortex, the electrical activity is associated with large neuronal Ca(2+) transients. Here we implemented an optogenetic approach to explore causal features of the generation of slow oscillation-associated Ca(2+) waves in the in vivo mouse brain. We demonstrate that brief optogenetic stimulation (3-20 ms) of a local group of layer 5 cortical neurons is sufficient for the induction of global brain Ca(2+) waves. These Ca(2+) waves are evoked in an all-or-none manner, exhibit refractoriness during repetitive stimulation, and propagate over long distances. By local optogenetic stimulation, we demonstrate that evoked Ca(2+) waves initially invade the cortex, followed by a secondary recruitment of the thalamus. Together, our results establish that synchronous activity in a small cluster of layer 5 cortical neurons can initiate a global neuronal wave of activity suited for long-range corticothalamic integration.

Sound-evoked network calcium transients in mouse auditory cortex in vivo.

Population calcium signals generated by the action potential activity of local clusters of neurons have been recorded in the auditory cortex of mice using an optical fibre-based approach. These network calcium transients (NCaTs) occurred spontaneously as well as in response to sound stimulation. Two-photon calcium imaging experiments suggest that neurons and neuropil contribute about equally to the NCaT. Sound-evoked calcium signals had two components: an early, fast increase in calcium concentration, which corresponds to the short-latency spiking responses observed in electrophysiological experiments, and a late, slow calcium transient which lasted for at least 1 s. The slow calcium transients evoked by sound were essentially identical to spontaneous NCaTs. Their sizes were dependent on the spontaneous activity level at sound onset, suggesting that spontaneous and sensory-evoked NCaTs excluded each other. When using pure tones as stimulus, the early evoked calcium transients were more narrowly tuned than the slow NCaTs. The slow NCaTs were correlated with global 'up states' recorded with epidural potentials, and sound presented during an epidural 'down state' triggered a calcium transient that was associated with an epidural 'up state'. Essentially indistinguishable calcium transients were evoked by optogenetic activation of local clusters of layer 5 pyramidal neurons in the auditory cortex, indicating that these neurons play an important role in the generation of the calcium signal. Taken together, our results identify sound-evoked slow NCaTs as an integral component of neuronal signalling in the mouse auditory cortex, reflecting the prolonged neuronal activity of local clusters of neurons that can be activated even by brief stimuli.

Formalin-induced fluorescence reveals cell shape and morphology in biological tissue samples.

Ultramicroscopy is a powerful tool to reveal detailed three-dimensional structures of large microscopical objects. Using high magnification, we observed that formalin induces fluorescence more in extra-cellular space and stains cellular structures negatively, rendering cells as dark objects in front of a bright background. Here, we show this effect on a three-dimensional image stack of a hippocampus sample, focusing on the CA1 region. This method, called FIF-Ultramicroscopy, allows for the three-dimensional observation of cellular structures in various tissue types without complicated staining techniques.

Infrared-guided laser stimulation of neurons in brain slices.

INTRODUCTIONThis protocol describes an approach for easy and precise stimulation of single neurons in brain slices using caged neurotransmitters. The technique can be applied to neurobiological problems for which precise and rapid stimulation of neurons in brain slices is required. When a caged neurotransmitter is added to the superfusion medium, neurons in brain slices can be excited by shining light on them. This very localized application allows dendrites to be scanned for the distribution of neurotransmitter receptors. Furthermore, by the stimulation of neighboring neurons, the connectivity of neuronal networks can be investigated. As laser stimulation can be performed quickly, this technique can also be used to search for synaptic connections between distant neurons with a low probability of connectivity.

Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.

Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

Corticotropin-releasing factor (CRF) receptor type 1-dependent modulation of synaptic plasticity.

CRF receptor type (CRHR) 1 exerts neuroregulatory control on associative learning processes such as fear and anxiety like behaviour. Using hippocampal slices, we investigated the neuronal excitability in mice lacking CRHR1 (Crhr1(-/-)). Compared to wild-type mice, long-term potentiation (LTP) elicited by 100 pulses at 100Hz was not different. Unexpectedly, at lower frequencies (1, 5 or 10Hz), the resulting synaptic changes in CA1 neurons of Crhr1(-/-) were systematically shifted towards long-term depression (LTD). Furthermore, testing paired-pulse paradigm revealed a GABA receptor-dependent decrease of paired-pulse ratio in Crhr1(-/-). It might be assumed that a lack of CRHR1 induce developmental changes which resulted in altered GABAergic activity, producing attenuated synaptic potentiation after repetitive stimulation and thus favouring LTD in principal neurons. Since CRHR1 are located in GABAergic somata, axons and boutons the activity of these receptor types rather might contribute to the development of the neuronal ability for plasticity like processes on the level of NMDAR subunit composition and GABAergic activity.

Cannabinoid receptor type 1 located on presynaptic terminals of principal neurons in the forebrain controls glutamatergic synaptic transmission.

It is widely accepted that cannabinoids regulate GABA release by activation of cannabinoid receptor type 1 (CB1). Results obtained from a variety of brain regions consistently indicate that cannabinoid agonists can also reduce glutamatergic synaptic transmission. However, there are still conflicting data concerning the role of CB1 in cannabinoid-induced inhibition of glutamatergic transmission in cortical areas. Here, we provide direct evidence that activation of CB1 on terminals of principal neurons controls excitatory synaptic responses in the forebrain. In slices of the basolateral amygdala, the CA1 region of the hippocampus, and the primary somatosensory cortex of wild-type mice, application of the CB1 agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55,212-2; WIN) (5 mum) reduced evoked excitatory postsynaptic responses. In contrast, in slices obtained from conditional mouse mutants lacking CB1 in all principal forebrain neurons but not in GABAergic interneurons (CB1(f/f;CaMKIIalphaCre)), WIN no longer affected glutamatergic synaptic transmission in any of the brain regions tested. Compatible with a presynaptic mechanism, WIN did not change the sensitivity to focally uncaged l-glutamate. WIN reduced glutamatergic responses in slices obtained from mice lacking CB1 exclusively in GABAergic neurons (CB1(f/f;Dlx5/6-Cre)), thus excluding the involvement of CB1 expressed on GABAergic neurons in this effect of the drug. The present data strongly indicate that excitatory synaptic transmission in forebrain areas is directly modulated by CB1 expressed on presynaptic axon terminals originating from glutamatergic neurons.

Neramexane (merz pharmaceuticals/forest laboratories).

Merz Pharmaceuticals GmbH and Forest Laboratories Inc are developing neramexane, an oral N-methyl-D-aspartate antagonist, as a potential neuroprotectant for various central nervous system disorders, including Alzheimer's disease, and for the potential treatment of drug and alcohol dependence, and pain.

Effects of sensory deprivation on columnar organization of neuronal circuits in the rat barrel cortex.

We examined whether sensory deprivation during formation of the cortical circuitry influences the pattern of intracortical single-cell connections in rat barrel cortex. Excitatory postsynaptic potentials (EPSPs) from layer 2/3 (L2/3) pyramidal neurons were recorded in vitro using patch-clamp techniques. In order to evoke EPSPs, presynaptic neurons were stimulated by photolytically applied glutamate, thus generating action potentials. Synaptic connections between the stimulated and the recorded neuron were identified by the occurrence of PSPs following photostimulation. Sensory deprivation changed the pattern of projections from L4 and L2/3 neurons to L2/3 pyramidal cells. In slices of non-deprived rats 86% of the total presynaptic neurons were located in the first and only 10% in the second barrel column. Deprivation changed these values to 67% and 26%, respectively. Therefore, the probability of presynaptic cells projecting to L2/3 neurons was shifted from adjacent to more remote barrel columns. These results indicate that deprivation of sensory input influences the pattern of intracortical connections.

Sensory deprivation changes the pattern of synaptic connectivity in rat barrel cortex.

We examined whether sensory deprivation during formation of the cortical circuitry influences the pattern of intracortical single-cell connections in rat barrel cortex. Excitatory postsynaptic potentials from layer 5 pyramidal neurons were recorded in vitro using patch-clamp techniques. In order to evoke such postsynaptic potentials presumptive presynaptic neurons were stimulated by photolytically applied glutamate thus generating action potentials. Synaptic connections between the stimulated and the recorded neuron were identified by the occurrence of postsynaptic potentials following photostimulation. Sensory deprivation altered the projections from layer 2/3 neurons to layer 5 pyramidal cells (L2/3-->L5 projections). In slices of non-deprived rats the input probability of L2/3-->L5 projections showed a periodic pattern with more synaptic connections originating from the borders of the barrel columns, and less synaptic connections originating from the centres. After whisker clipping this periodic pattern disappeared completely and the input probability declined monotonically with increasing distance between stimulated and recorded neuron. These results indicate that sensory input is a prerequisite to establish a synaptic projection pattern which is correlated to the columnar organisation of the anatomical barrel structure.

Distribution and properties of functional postsynaptic kainate receptors on neocortical layer V pyramidal neurons.

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic transmission and signal processing. In the present study we investigated the location and properties of functional kainate receptors (KARs) on the somatodendritic membrane of rat neocortical layer V pyramidal neurons. Infrared-guided laser stimulation was used to apply glutamate photolytically to the soma and various sites along the apical dendrite. Electrical currents, resulting from the activation of pharmacologically isolated KARs, were measured by whole-cell patch-clamp recording. In addition, KARs on somatic and dendritic outside-out patches were activated while still within the brain tissue. We found that functional KARs are located on the entire somatodendritic membrane that was examined. Fast kinetics, a linear I-V relationship, and a relatively high single-channel conductance are characteristic features of these receptors. We provide evidence that the unitary properties of somatic and dendritic KARs are identical. Regarding the subcellular distribution of KARs, our results indicate that the density of these receptors increases toward the distal dendrite. They are located mainly at extrasynaptic sites but also mediate fast synaptic signaling triggered by afferent stimulation. The differential distribution speaks in favor of a selective targeting of KARs on central neurons and may reflect a mechanism for a location-dependent regulation of synaptic efficacy. Furthermore, it is feasible to assume that extrasynaptic KARs could be activated by a "spillover" of synaptically released glutamate, ambient glutamate in the CSF, or glutamate released from adjacent astrocytes.

Infrared-guided laser stimulation of neurons in brain slices.

Infrared-guided laser stimulation is a new technique that allows precise and rapid stimulation of visualized neurons in brain slices. Infrared imaging of neurons with a new contrast system is combined with the photolytic release of caged neurotransmitters by an ultraviolet (UV) laser. Addition of caged neurotransmitters to the superfusion medium of neurons in brain slices allows local excitation in the micrometer range with a focused spot of UV light. In this way, the distribution of glutamate or gamma-aminobutyric acid (GABA) receptors on neuronal dendrites can be mapped. Furthermore, this technique can be used to map the connectivity of neuronal networks through the controlled stimulation of neighboring neurons. Because the laser stimulation can be performed much faster than can paired recording, it is also possible to search for synaptic connections between distant neurons that have a low probability of connectivity.