RESUMEN
We used whole cell recordings from slice preparations of mouse cortex to identify various inputs to neurons of layer 1. Two sensory cortical areas were targeted: a primary somatosensory area, namely, the barrel cortex of S1, and a higher order visual area, namely, V2M. Results were similar from both areas. By activating local inputs using photostimulation with caged glutamate, we also identified glutamatergic (and possibly GABAergic) inputs from all lower layers plus GABAergic inputs from nearby layer 1 neurons. However, the patterns of such inputs to layer 1 neurons showed great variation among cells. In separate experiments, we found that electrical stimulation of axons running parallel to the cortical surface in layer 1 also evoked a variety of convergent input types to layer 1 neurons, including glutamatergic "drivers" and "modulators" plus classic modulatory inputs, including serotonergic, nicotinic, α- and ß-adrenergic, from subcortical sites. Given that these layer 1 cells significantly affect the responses of other cortical neurons, especially via affecting the apical dendrites of pyramidal cells so important to cortical functioning, their role in cortical processing is significant. We believe that the data presented here lead to better understanding of the functioning of layer 1 neurons in their role of influencing cortical processing.
Asunto(s)
Neuronas/fisiología , Corteza Somatosensorial/fisiología , Animales , Estimulación Eléctrica , Femenino , Masculino , Ratones , Vías Nerviosas , Técnicas de Placa-ClampRESUMEN
The thalamic reticular nucleus (TRN) is a thin layer of GABAergic cells lying rostral and lateral to the dorsal thalamus, and its projection to thalamic relay cells (i.e., the reticulothalamic pathway) strongly inhibits these cells. In an attempt to extend earlier studies of reticulothalamic connections to sensory thalamic nuclei, we used laser-scanning photostimulation to study the reticulothalamic projections to the main motor thalamic relays, the ventral anterior and lateral (VA and VL) nuclei, as well as to the nearby central lateral (CL) thalamic nucleus. VA/VL and the earlier studied somatosensory thalamic nuclei are considered "core" nuclei with topographic thalamocortical projections, whereas CL is thought to be a "matrix" nucleus with diffuse thalamocortical projections. We found that the TRN input footprints to VA/VL and CL are spatially localized and topographic and generally conform to the patterns established earlier for the TRN projections to sensory thalamic relays. These remarkable similarities suggest similar organization of reticulothalamic pathways and TRN regulation of thalamocortical communication for motor and sensory systems and perhaps also for core and matrix thalamus. Furthermore, we found that VA/VL and CL shared overlapping TRN input regions, suggesting that CL may also be involved in the relay of motor information.
Asunto(s)
Mapeo Encefálico , Vías Eferentes/anatomía & histología , Vías Eferentes/fisiología , Núcleos Talámicos/citología , Núcleos Talámicos/fisiología , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Femenino , Procesamiento de Imagen Asistido por Computador , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Neuronas/fisiología , Técnicas de Placa-Clamp , Estimulación LuminosaRESUMEN
The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.
Asunto(s)
Red Nerviosa/fisiología , Estimulación Luminosa/métodos , Prosencéfalo/fisiología , Adenoviridae/genética , Animales , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Microscopía Confocal , Neuronas/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Rodopsina/biosíntesis , Rodopsina/química , Rodopsina/genética , Sinapsis/fisiología , TransfecciónRESUMEN
Axonal branches from a subset of neurons in cerebral cortical layer 6 innervate both cortical layer 4 and the thalamus. As such, these neurons are poised to modulate thalamocortical transmission at multiple forebrain sites. Here, we examined the functional organization of the layer 6 intracortical projections in auditory, somatosensory, and visual cortical areas using an optogenetic approach to specifically target these neurons. We characterized the anatomical and physiological organization of these projections using laser-scanning photostimulation to functionally map the elicited postsynaptic responses in layer 4. We found that these responses originated from regions over 1 mm in width, eliciting short-term facilitating responses. These results indicate that intracortical modulation of layer 4 occurs through widespread layer 6 projections in each sensory cortical area.
Asunto(s)
Corteza Auditiva/fisiología , Neuronas/fisiología , Tálamo/fisiología , Corteza Visual/fisiología , Animales , Mapeo Encefálico , Ratones , Ratones Transgénicos , Vías Nerviosas/fisiología , Corteza Somatosensorial/fisiologíaRESUMEN
Most axons connecting the thalamus and cortex in both directions pass through the thalamic reticular nucleus (TRN), a thin layer of GABAergic cells adjacent to the thalamus, and innervate neurons there. The TRN, therefore, is in a strategic location to regulate thalamocortical communication. We recorded neurons of the somatosensory region of the TRN in a thalamocortical slice preparation and studied the spatial organization of their thalamic input using laser scanning photostimulation. We show that the thalamoreticular pathway is organized topographically for most neurons. The somatosensory region of the TRN can be organized into three tiers. From the inner (thalamoreticular) border to the outer, in a manner roughly reciprocal to the reticulothalamic pathway, each of these tiers receives its input from one of the somatosensory relays of the thalamus--the posterior medial, ventroposterior medial, and ventroposterior lateral nuclei. What is surprising is that approximately a quarter of the recorded neurons received input from multiple thalamic regions usually located in different nuclei. These neurons distribute evenly throughout the thickness of the TRN. Our results, therefore, suggest that there exist a subpopulation of TRN neurons that receive convergent inputs from multiple thalamic sources and engage in more complex patterns of inhibition of relay cells. We propose these neurons enable the TRN to act as an externally driven "searchlight" that integrates cortical and subcortical inputs and then inhibits or disinhibits specific thalamic relay cells, so that appropriate information can get through the thalamus to the cortex.
Asunto(s)
Axones/fisiología , Neuronas/fisiología , Núcleos Talámicos/fisiología , Tálamo/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Vías Nerviosas/fisiología , Técnicas de Placa-ClampRESUMEN
The pathway from cortical layer 6 to the thalamus is a property of all thalamic relay nuclei. This pathway, as a population, directly excites relay cells and indirectly inhibits them via the thalamic reticular nucleus. To understand the circuit organization of this cortical feedback, we used laser-scanning photostimulation, which specifically activates somata or dendrites, to stimulate the primary somatosensory cortex in an in vitro thalamocortical slice preparation while recording from neurons of the ventral posterior medial nucleus. Layer 6 photostimulation evoked biphasic excitatory postsynaptic current/inhibitory postsynaptic current (EPSC/IPSC) responses in the neurons of the ventral posterior medial nucleus, indicating that such photostimulation strongly activates reticular cells. These disynaptic IPSCs were greatly suppressed or abolished by bath application of the muscarinic agonist acetyl-beta-methylcholine. Our results suggest that the top-down modulation of thalamic neurons from cortical layer 6 involves an inhibitory component via the thalamic reticular nucleus, and this component can be selectively reduced by cholinergic input. Finally, we found the footprints for the excitatory corticothalamic and the inhibitory cortico-reticulo-thalamic inputs to be located in similar positions, though in some cases they are offset. Both patterns have implications for cortico-reticulo-thalamic circuitry.
Asunto(s)
Fibras Colinérgicas/fisiología , Potenciales Postsinápticos Inhibidores/fisiología , Vías Nerviosas/fisiología , Corteza Somatosensorial/fisiología , Núcleos Talámicos Ventrales/fisiología , Animales , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Cloruro de Metacolina/farmacología , Ratones , Agonistas Muscarínicos/farmacologíaRESUMEN
The thalamic reticular nucleus is a layer of GABAergic neurons that occupy a strategic position between the thalamus and cortex. Here we used laser scanning photostimulation to compare in young mice (9-12 days old) the organization of the reticular inputs to first- and higher-order somatosensory relays, namely, the ventral posterior lateral nucleus and posterior nucleus, respectively. The reticulothalamic input footprints to the ventral posterior lateral nucleus neurons consisted of small, single, topographically organized elliptical regions in a tier away from the reticulothalamic border. In contrast, those to the posterior nucleus were complicated and varied considerably among neurons: although almost all contained a single elliptical region near the reticulothalamic border, in most cases, they consisted of additional discontinuous regions or relatively diffuse regions throughout the thickness of the thalamic reticular nucleus. Our results suggest two sources of reticular inputs to the posterior nucleus neurons: one that is relatively topographic from regions near the reticulothalamic border and one that is relatively diffuse and convergent from most or all of the thickness of the thalamic reticular nucleus. We propose that the more topographic reticular input is the basis of local inhibition seen in posterior nucleus neurons and that the more diffuse and convergent input may represent circuitry through which the ventral posterior lateral and posterior nuclei interact.
Asunto(s)
Mapeo Encefálico , Vías Nerviosas/fisiología , Neuronas/fisiología , Corteza Somatosensorial/fisiología , Núcleos Talámicos/fisiología , Animales , Animales Recién Nacidos , Técnicas In Vitro , Ratones , Ratones Endogámicos BALB C , Estimulación Luminosa/métodos , Núcleos Talámicos/citologíaRESUMEN
The thalamic reticular nucleus is strategically located in the axonal pathways between thalamus and cortex, and reticular cells exert strong, topographic inhibition on thalamic relay cells. Although evidence exists that reticular neurons are interconnected through conventional and electrical synapses, the spatial extent and relative strength of these synapses are unclear. To address these issues, we used uncaging of glutamate by laser-scanning photostimulation to provide precisely localized and consistent activation of reticular cell bodies and dendrites in an in vitro slice preparation from the rat as a means to study reticulo-reticular connections. Among the 47 recorded reticular neurons, 29 (62%) received GABAergic axodendritic input from an area immediately surrounding each of the recorded cell bodies, and 8 (17%) responded with depolarizing spikelets, suggesting inputs through electrical synapses. We also found that TTX completely blocked all evoked IPSCs, implying that any dendrodendritic synapses between reticular cells either are relatively weak, have no nearby glutamatergic receptors, or are dependent on back-propagation of action potentials. Finally, we showed that the GABAergic connections between reticular cells are weaker than those from reticular cells to relay cells. Our results suggest that the GABAergic axodendritic synapse is the dominant form of reticulo-reticular connectivity, and because they are much weaker than the reticulo-relay cell synapses, their functional purpose may be to regulate the spatial extent of the reticular inhibition on relay cells.
Asunto(s)
Interneuronas/fisiología , Formación Reticular/fisiología , Núcleos Talámicos/fisiología , Animales , Axones/fisiología , Mapeo Encefálico , Interpretación Estadística de Datos , Electrofisiología , Potenciales Postsinápticos Excitadores/fisiología , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/fisiología , Ácido Glutámico/metabolismo , Técnicas In Vitro , Rayos Láser , Vías Nerviosas/fisiología , Estimulación Luminosa , Piridazinas/farmacología , Ratas , Formación Reticular/citología , Sinapsis/fisiología , Tetrodotoxina/farmacología , Núcleos Talámicos/citología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
We used laser scanning photostimulation through a focused UV laser of caged glutamate in an in vitro slice preparation through the rat's somatosensory thalamus to study topography and connectivity between the thalamic reticular nucleus and ventral posterior lateral nucleus. This enabled us to focally stimulate the soma or dendrites of reticular neurons. We were thus able to confirm and extend previous observations based mainly on neuroanatomical pathway tracing techniques: the projections from the thalamic reticular nucleus to the ventral posterior lateral nucleus have precise topography. The reticular zone, which we refer to as a "footprint," within which photostimulation evoked inhibitory postsynaptic currents (IPSCs) in relay cells, was relatively small and oval, with the long axis being parallel to the border between the thalamic reticular nucleus and ventral posterior lateral nucleus. These evoked IPSCs were large, and by using appropriate GABA antagonists, we were able to show both GABA(A) and GABA(B) components. This suggests that photostimulation strongly activated reticular neurons. Finally, we were able to activate a disynaptic relay cell-to-reticular-to- relay cell pathway by evoking IPSCs in relay cells from photostimulation of the region surrounding a recorded relay cell. This, too, suggests strong responses of relay cells, responses strong enough to evoke spiking in their postsynaptic reticular targets. The regions of photostimulation for these disynaptic responses were much larger than the above-mentioned reticular footprints, and this suggests that reticulothalamic axon arbors are less widespread than thalamoreticular arbors, that there is more convergence in thalamoreticular connections than in reticulothalamic connections, or both.
Asunto(s)
Mapeo Encefálico , Núcleos Talámicos Intralaminares/citología , Núcleos Talámicos Laterales/citología , Vías Nerviosas/fisiología , Neuronas/efectos de la radiación , Estimulación Luminosa/métodos , Animales , Animales Recién Nacidos , Interacciones Farmacológicas , Antagonistas del GABA/farmacología , Técnicas In Vitro , Núcleos Talámicos Intralaminares/fisiología , Rayos Láser , Núcleos Talámicos Laterales/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Inhibición Neural/efectos de la radiación , Vías Nerviosas/anatomía & histología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Ácidos Fosfínicos/farmacología , Estimulación Luminosa/instrumentación , Piridazinas/farmacología , RatasRESUMEN
The odour-induced population response in the in vivo turtle (Terepene sp.) olfactory bulb consists of three oscillatory components (rostral, middle and caudal) that ride on top of a DC signal. In an initial step to determine the functional role of these four signals, we compared the signals elicited by different odorants. Most experiments compared isoamyl acetate and cineole, odorants which have very different maps of input to olfactory bulb glomeruli in the turtle and a different perceptual quality for humans. We found substantial differences in the response to the two odours in the rise-time of the DC signal and in the latency of the middle oscillation. The rate of rise for cineole was twice as fast as that for isoamyl acetate. Similarly, the latency for the middle oscillation was about twice as long for isoamyl acetate as it was for cineole. On the other hand, a number of characteristics of the signals were not substantially different for the two odorants. These included the latency of the rostral and caudal oscillation, the frequency and envelope of all three oscillations and their locations and spatial extents. A smaller number of experiments were carried out with hexanone and hexanal; the oscillations elicited by these odorants did not appear to be different from those elicited by isoamyl acetate and cineole. Qualitative differences between the oscillations in the turtle and those in two invertebrate phyla suggest that different odour processing strategies may be used.