RESUMEN
Breathing requires distinct patterns of neuronal activity in the brainstem. The most critical part of the neuronal network responsible for respiratory rhythm generation is the preBötzinger Complex (preBötC), located in the ventrolateral medulla. This area contains both rhythmogenic glutamatergic neurons and also a high number of inhibitory neurons. Here, we aimed to analyze the activity of glycinergic neurons in the preBötC in anesthetized mice. To identify inhibitory neurons, we used a transgenic mouse line that allows expression of Channelrhodopsin 2 in glycinergic neurons. Using juxtacellular recordings and optogenetic activation via a single recording electrode, we were able to identify neurons as inhibitory and define their activity pattern in relation to the breathing rhythm. We could show that the activity pattern of glycinergic respiratory neurons in the preBötC was heterogeneous. Interestingly, only a minority of the identified glycinergic neurons showed a clear phase-locked activity pattern in every respiratory cycle. Taken together, we could show that neuron identification is possible by a combination of juxtacellular recordings and optogenetic activation via a single recording electrode.
Asunto(s)
Optogenética , Centro Respiratorio , Ratones , Animales , Centro Respiratorio/fisiología , Neuronas/metabolismo , Bulbo Raquídeo/fisiología , Ratones TransgénicosRESUMEN
Neural circuits are made of a vast diversity of neuronal cell types. While immense progress has been made in classifying neurons based on morphological, molecular, and functional properties, understanding how this heterogeneity contributes to brain function during natural behavior has remained largely unresolved. In the present study, we combined the juxtacellular recording and labeling technique with optogenetics in freely moving mice. This allowed us to selectively target molecularly defined cell classes for in vivo single-cell recordings and morphological analysis. We validated this strategy in the CA1 region of the mouse hippocampus by restricting Channelrhodopsin expression to Calbindin-positive neurons. Directly versus indirectly light-activated neurons could be readily distinguished based on the latencies of light-evoked spikes, with juxtacellular labeling and post hoc histological analysis providing 'ground-truth' validation. Using these opto-juxtacellular procedures in freely moving mice, we found that Calbindin-positive CA1 pyramidal cells were weakly spatially modulated and conveyed less spatial information than Calbindin-negative neurons - pointing to pyramidal cell identity as a key determinant for neuronal recruitment into the hippocampal spatial map. Thus, our method complements current in vivo techniques by enabling optogenetic-assisted structure-function analysis of single neurons recorded during natural, unrestrained behavior.
Asunto(s)
Región CA1 Hipocampal/fisiología , Hipocampo/metabolismo , Movimiento/fisiología , Neuronas/fisiología , Células Piramidales/fisiología , Potenciales de Acción/fisiología , Animales , Región CA1 Hipocampal/química , Calbindinas/genética , Channelrhodopsins/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Optogenética/métodos , Células Piramidales/químicaRESUMEN
Nicotine stimulates dopamine (DA) neurons of the ventral tegmental area (VTA) to establish and maintain reinforcement. Nicotine also induces anxiety through an as yet unknown circuitry. We found that nicotine injection drives opposite functional responses of two distinct populations of VTA DA neurons with anatomically segregated projections: it activates neurons that project to the nucleus accumbens (NAc), whereas it inhibits neurons that project to the amygdala nuclei (Amg). We further show that nicotine mediates anxiety-like behavior by acting on ß2-subunit-containing nicotinic acetylcholine receptors of the VTA. Finally, using optogenetics, we bidirectionally manipulate the VTA-NAc and VTA-Amg pathways to dissociate their contributions to anxiety-like behavior. We show that inhibition of VTA-Amg DA neurons mediates anxiety-like behavior, while their activation prevents the anxiogenic effects of nicotine. These distinct subpopulations of VTA DA neurons with opposite responses to nicotine may differentially drive the anxiogenic and the reinforcing effects of nicotine.
Asunto(s)
Ansiedad/tratamiento farmacológico , Vías Nerviosas/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Área Tegmental Ventral/efectos de los fármacos , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Ansiedad/inducido químicamente , Ansiedad/fisiopatología , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Masculino , Ratones , Vías Nerviosas/fisiología , Nicotina/metabolismo , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/fisiología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Refuerzo en Psicología , Área Tegmental Ventral/fisiologíaRESUMEN
Plasticity within hippocampal circuits is essential for memory functions. The hippocampal CA2/CA3 region is thought to be able to rapidly store incoming information by plastic modifications of synaptic weights within its recurrent network. High-frequency spike-bursts are believed to be essential for this process, by serving as triggers for synaptic plasticity. Given the diversity of CA2/CA3 pyramidal neurons, it is currently unknown whether and how burst activity, assessed in vivo during natural behavior, relates to principal cell heterogeneity. To explore this issue, we juxtacellularly recorded the activity of single CA2/CA3 neurons from freely-moving male mice, exploring a familiar environment. In line with previous work, we found that spatial and temporal activity patterns of pyramidal neurons correlated with their topographical position. Morphometric analysis revealed that neurons with a higher proportion of distal dendritic length displayed a higher tendency to fire spike-bursts. We propose that the dendritic architecture of pyramidal neurons might determine burst-firing by setting the relative amount of distal excitatory inputs from the entorhinal cortex.SIGNIFICANCE STATEMENT High-frequency spike-bursts are thought to serve fundamental computational roles within neural circuits. Within hippocampal circuits, spike-bursts are believed to serve as potent instructive signals, which increase the efficiency of information transfer and induce rapid modifications of synaptic efficacies. In the present study, by juxtacellularly recording and labeling single CA2/CA3 neurons in freely-moving mice, we explored whether and how burst propensity relates to pyramidal cell heterogeneity. We provide evidence that, within the CA2/CA3 region, neurons with higher proportion of distal dendritic length display a higher tendency to fire spike-bursts. Thus, the relative amount of entorhinal inputs, arriving onto the distal dendrites, might determine the burst propensity of individual CA2/CA3 neurons in vivo during natural behavior.
Asunto(s)
Región CA2 Hipocampal/fisiología , Región CA3 Hipocampal/fisiología , Movimiento/fisiología , Células Piramidales/fisiología , Potenciales de Acción/fisiología , Animales , Región CA2 Hipocampal/química , Región CA3 Hipocampal/química , Masculino , Ratones , Ratones Endogámicos C57BL , Células Piramidales/químicaRESUMEN
BACKGROUND: Understanding the configuration of neural circuits and the specific role of distinct cortical neuron types involved in behavior, requires the study of structure-function and connectivity relationships with single cell resolution in awake behaving animals. Despite head-fixed behaving rats have been used for in vivo measuring of neuronal activity, it is a concern that head fixation could change the performance of behavioral task. NEW METHOD: We describe the procedures for efficiently training Wistar rats to develop a behavioral task, involving planning and execution of a qualified movement in response to a visual cue under head-fixed conditions. The behavioral and movement performance in freely moving vs head-fixed conditions was analyzed. RESULTS: The best behavioral performance was obtained in the rats that were trained first in freely moving conditions and then placed in a head-restrained condition compared with the animals which first were habituated to head-restriction and then learned the task. Moreover, head restriction did not alter the movement performance. Stable juxtacellular recordings from sensorimotor cortex neurons were obtained while the rats were performing forelimb movements. Biocytin electroporation and retrograde tracer injections, permits identify the hodology of individual long-range projecting neurons. COMPARISON WITH EXISTING METHODS: Our method shows no difference in the behavioral performance of head fixed and freely moving conditions. Also includes a computer aided design of a discrete and ergonomic head-post allowing enough stability to perform juxtacellular recording and labeling of cortical neurons. CONCLUSIONS: Our method is suitable for the in vivo characterization of neuronal circuits and their long-range connectivity.
Asunto(s)
Conducta Animal/fisiología , Condicionamiento Operante/fisiología , Conectoma/métodos , Electrocorticografía/métodos , Actividad Motora/fisiología , Neuronas/fisiología , Restricción Física , Corteza Sensoriomotora/fisiología , Animales , Electroporación , Miembro Anterior/fisiología , Movimientos de la Cabeza/fisiología , Técnicas de Trazados de Vías Neuroanatómicas , Desempeño Psicomotor/fisiología , Ratas , Ratas WistarRESUMEN
Layer 3 of the medial entorhinal cortex is a major gateway from the neocortex to the hippocampus. Here we addressed structure-function relationships in medial entorhinal cortex layer 3 by combining anatomical analysis with juxtacellular identification of single neurons in freely behaving rats. Anatomically, layer 3 appears as a relatively homogeneous cell sheet. Dual-retrograde neuronal tracing experiments indicate a large overlap between layer 3 pyramidal populations, which project to ipsilateral hippocampus, and the contralateral medial entorhinal cortex. These cells were intermingled within layer 3, and had similar morphological and intrinsic electrophysiological properties. Dendritic trees of layer 3 neurons largely avoided the calbindin-positive patches in layer 2. Identification of layer 3 neurons during spatial exploration (n = 17) and extracellular recordings (n = 52) pointed to homogeneous spatial discharge patterns. Layer 3 neurons showed only weak spiking theta rhythmicity and sparse head-direction selectivity. A majority of cells (50 of 69) showed no significant spatial modulation. All of the â¼28% of neurons that carried significant amounts of spatial information (19 of 69) discharged in irregular spatial patterns. Thus, layer 3 spatiotemporal firing properties are remarkably different from those of layer 2, where theta rhythmicity is prominent and spatially modulated cells often discharge in grid or border patterns. Significance statement: Neurons within the superficial layers of the medial entorhinal cortex (MEC) often discharge in border, head-direction, and theta-modulated grid patterns. It is still largely unknown how defined discharge patterns relate to cellular diversity in the superficial layers of the MEC. In the present study, we addressed this issue by combining anatomical analysis with juxtacellular identification of single layer 3 neurons in freely behaving rats. We provide evidence that the anatomical organization and spatiotemporal firing properties of layer 3 neurons are remarkably different from those in layer 2. Specifically, most layer 3 neurons discharged in spatially irregular firing patterns, with weak theta-modulation and head-directional selectivity. This work thus poses constraints on the spatiotemporal patterns reaching downstream targets, like the hippocampus.