Topography and response timing of intact cerebellum stained with absorbance voltage-sensitive dye.
ABSTRACT: Physiological activity of the turtle cerebellar cortex (Cb), maintained in vitro, was recorded during microstimulation of inferior olive (IO). Previous single-electrode responses to such stimulation showed similar latencies across a limited region of Cb, yet those recordings lacked spatial and temporal resolution and the recording depth was variable. The topography and timing of those responses were reexamined using photodiode optical recordings. Because turtle Cb is thin and unfoliated, its entire surface can be stained by a voltage-sensitive dye and transilluminated to measure changes in its local absorbance. Microstimulation of the IO evoked widespread depolarization from the rostral to the caudal edge of the contralateral Cb. The time course of responses measured at a single photodiode matched that of single-microelectrode responses in the corresponding Cb locus. The largest and most readily evoked response was a sagittal band centered about 0.7 mm from the midline. Focal white-matter (WM) microstimulation on the ventricular surface also activated sagittal bands, whereas stimulation of adjacent granule cells evoked a radial patch of activation. In contrast, molecular-layer (ML) microstimulation evoked transverse beams of activation, centered on the rostrocaudal stimulus position, which traveled bidirectionally across the midline to the lateral edges of the Cb. A timing analysis demonstrated that both IO and WM microstimulation evoked responses with a nearly simultaneous onset along a sagittal band, whereas ML microstimulation evoked a slowly propagating wave traveling about 25 cm/s. The response similarity to IO and WM microstimulation suggests that the responses to WM microstimulation are dominated by activation of its climbing fibers. The Cb's role in the generation of precise motor control may result from these temporal and topographic differences in orthogonally oriented pathways. Optical recordings of the turtle's thin flat Cb can provide insights into that role.
Project description:Optical recording techniques were applied to the turtle cerebellum to localize synchronous responses to microstimulation of its cortical layers and reveal the cerebellum's three-dimensional processing. The in vitro yet intact cerebellum was first immersed in voltage-sensitive dye and its responses while intact were compared to those measured in thick cerebellar slices. Each slice is stained throughout its depth, even though the pial half appeared darker during epi-illumination and lighter during trans-illumination. Optical responses were shown to be mediated by the voltage-sensitive dye because the evoked signals had opposite polarity for 540- and 710-nm light, but no response to 850-nm light. Molecular layer stimulation of the intact cerebellum evoked slow transverse beams. Similar beams were observed in the molecular layer of thick transverse slices but not sagittal slices. With low currents, beams in transverse slices were restricted to sublayers within the molecular layer, conducting slowly away from the stimulus site. These excitatory beams were observed nearly all the way across the turtle cerebellum, distances of 4-6mm. Microstimulation of the granule cell layer of both transverse or sagittal slices evoked a local membrane depolarization restricted to a radial wedge, but these radial responses did not activate measurable molecular layer beams in transverse slices. White matter microstimulation in sagittal slices (near the ventricular surface of the turtle cerebellum) activated the granule cell and Purkinje cell layers, but not the molecular layer. These responses were nearly synchronous, were primarily caudal to the stimulation, and were blocked by cobalt ions. Therefore, synaptic responses in all cerebellar layers contribute to optical signals recorded in intact cerebellum in vitro (Brown and Ariel, 2009). Rapid radial signaling connects a sagittally-oriented, fast-conduction system of the deep layers with the transverse-oriented, slow-conducting molecular layer, thereby permitting complex temporal processing between two tangential but orthogonal paths in the cerebellar cortex.
Project description:Voltage-sensitive dye activity within the thin, unfoliated turtle cerebellar cortex (Cb) was recorded in vitro during eighth cranial nerve (nVIII) stimulation. Short latency responses were localized to the middle of the lateral edges of both ipsilateral and contralateral Cb [vestibulocerebellum (vCb)]. Even with a severed contralateral Cb peduncle, stimulation of the nVIII ipsilateral to the intact peduncle evoked contralateral vCb responses with a mean latency of only 0.25 ms after the ipsilateral responses, even though the distance between them was ? 5 mm. We investigated whether a rapidly conducting commissure exists between each vCb by stimulating one of them directly. Responses in both vCb spread sagittally, but, surprisingly, there was no sequential activation along a transverse Cb beam between them. In contrast, stimulation medial to either vCb evoked transverse beams that required ? 20 ms to cross the Cb. Therefore, the rapid commissural connection between each vCb is not mediated by slowly conducting parallel fibers. Also, the vCb was not strongly activated by climbing fiber stimulation, suggesting that inputs to vCb involve distinct cerebellar circuits. Responses between the two vCb remained following knife cuts through the rostral and caudal Cb along the midline, through both peduncles, and even shallow midline cuts to the middle Cb through its white matter and granule cell layer. Commissural responses were still observed only with a narrow transverse bridge between each vCb or in thick transverse Cb slices. Horseradish peroxidase transport from one vCb labeled transverse axons traveling within the Purkinje cell layer that were larger than parallel fibers and lacked varicosities. In sagittal sections, cross-section profiles of myelinated axons were observed around Purkinje cells midway between the rostral and caudal Cb. This novel pathway for transverse communication between lateral edges of turtle Cb suggests that afferents may directly conduct vestibular information rapidly across the Cb to coordinate vestibulomotor reflex behaviors.
Project description:In neurophysiology researches, peripheral stimulation is used along with recordings of neural activities to study the processing of somatosensory signals in the brain. However, limited precision of peripheral stimulation makes it difficult to activate the neuron with millisecond resolution and study its functional properties in this scale. Also, tissue/receptor damage that could occur in some experiments often limits the amount of responses that can be recorded and hence reduces data reproducibility. To overcome these limitations, electrical microstimulation (ES) of the brain could be used to directly and more precisely evoke neural responses. For this purpose, a deep-brain ES protocol for rat somatosensory relay neurons was developed in this study. Three male Wistar rats were used in the experiment. The ES was applied to the thalamic region responsive to hindpaw tactile stimulation (TS) via a theta glass microelectrode. The resulting ES-evoked cortical responses showed action potentials and thalamocortical relay latencies very similar to those evoked by TS. This result shows that the developed deep-brain ES protocol is an effective tool to bypass peripheral tissue for in vivo functional analysis of specific types of somatosensory neurons. This protocol could be readily applied in researches of nociception and other somatosensory systems to allow more extensive exploration of the neural functional networks.
Project description:We have previously demonstrated antinociceptive effects of fatty acid amide hydrolase (FAAH) inhibition that were accompanied by increases in the levels of endocannabinoids (ECs) in the hind paw. Here, the effects of the FAAH inhibitor URB597 (3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) on responses of spinal neurons were studied.Extracellular single-unit recordings of dorsal horn neurons were made in anaesthetized rats with hind paw inflammation induced by lambda-carrageenan. Effects of intraplantar pre-administration of URB597, or vehicle, on carrageenan-evoked expansion of peripheral receptive fields of spinal neurons and mechanically evoked responses of neurons were studied. The cannabinoid receptor type 1 (CB(1)) antagonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the peroxisome proliferator-activated receptor (PPAR)-alpha antagonist GW6471 ([(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxa zolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester) were used to investigate the roles of these receptors in mediating the effects of URB597.URB597 (25 microg in 50 microL) pretreatment significantly inhibited carrageenan-evoked receptive field expansion and this was significantly reversed by co-administration of the PPAR-alpha antagonist but not the CB(1) antagonist. Pretreatment with the PPAR-alpha receptor agonist WY14643 ([[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid) also significantly inhibited receptive field expansion. URB597 (25 or 100 microg in 50 microL) had no significant effect on mechanically evoked responses of spinal neurons.URB597 inhibited receptive field expansions but not mechanically evoked responses of spinal neurons in rats with hind paw inflammation. These effects were blocked by PPAR-alpha receptor antagonism. These data support the contention that URB597 exerts its antinociceptive effects by indirect inhibition of sensitization of neuronal responses at least partly through PPAR-alpha activation due to enhanced EC levels.
Project description:Deep brain stimulation (DBS) could become a palliative treatment for patients with drug-resistant epilepsy for which surgery cannot be proposed. The objective of this study was to perform microstimulation to measure the effects of DBS in epilepsy locally at the level of a few neurons, with microelectrode recordings, for the first time in patients with epilepsy. Microelectrode recordings were performed before, during and after microstimulation in nine patients with refractory epilepsy. Neuronal spikes were successfully extracted from multi-unit recordings with clustering in six out of seven patients during hippocampal and in one out of two patients during cortical dysplasia microstimulation (1 Hz, charge-balanced biphasic waveform, 60 ?s/ph, 25 ?A). The firing rates increased in four out of the six periods of microstimulation that could be analyzed. The firing rates were found higher than before microstimulation in all eight periods with increases reaching significance in six out of eight periods. Low-frequency microstimulation was hence sufficient to induce neuronal excitation lasting beyond the stimulation period. No inhibition was observed. This report presents the first evidence that microstimulation performed in epileptic patients produced locally neuronal excitation. Hence neuronal excitation is shown here as the local mechanism of action of DBS. This local excitation is in agreement with epileptogenic effects of low-frequency hippocampal macrostimulation.
Project description:Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception.We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo.Capsaicin (1-10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB? and metabotropic glutamate (mGlu?) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into 2-arachidonoylglycerol (2-AG), an endocannabinoid. Capsaicin induced membrane depolarization in 2/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB? and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist.Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu? receptors, yielding 2-AG from DAG by DAGL hydrolysis. 2-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB? receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.
Project description:Obesity has become a serious public health problem. Although diet, surgery, and exercise are the primary treatments for obesity, these activities are often supplemented using appetite suppressants. A previous study reported that obesity specialists frequently prescribed a new drug combination for its treatment that includes phentermine (Phen; dopaminergic appetite suppressant), a serotonin (5-HT) precursor 5-hydroxytryptophan (5-HTP; an appetite suppressant that increases the 5-HT concentration), and carbidopa (CB; peripheral blocker of conversion of 5-HTP to 5-HT). Despite its widespread use, there is neither a preclinical study confirming the drug efficacy nor studies of its effects on the brain. To fill this gap, in rats for seven consecutive days, we administered Phen intraperitoneally at different doses either alone or in combination with a fixed dose of 5-HTP/CB. In a different group, we infused drugs via an intraperitoneal catheter while extracellular-recordings were performed in the nucleus accumbens shell (NAcSh), a brain region with dopamine-releasing effects that is involved in the action of appetite suppressants. We found that the triple-drug combination leads to greater weight-loss than each drug alone. Moreover, and as the treatment progresses, the triple drug combination partially reversed psychomotor side-effects induced by Phen. Electrophysiological results revealed that Phen alone evoked a net inhibitory imbalance in NAcSh population activity that correlated with the onset of psychomotor effects. In addition, and unlike the greater weight loss, the addition of 5-HTP/CB did not alter the Phen-evoked inhibitory imbalance in NAcSh responses. Subsequent experiments shed light on the underlying mechanism. That is the majority of NAcSh neurons modulated by 5-HTP/CB were suppressed by Phen. Notably, and despite acting via a different mechanism of action (DA for Phen vs. 5-HT for 5-HTP/CB), both drugs recruited largely overlapping NAcSh neuronal ensembles. These data suggest that the neural correlates of the greater weight loss could be located outside the NAcSh, in other brain circuits. Furthermore, we conclude that Phen + 5-HTP/CB is a potential treatment for overweight and obesity.
Project description:Odors are initially encoded in the brain as a set of distinct physicochemical characteristics but are ultimately perceived as a unified sensory object--a "smell." It remains unclear how chemical features encoded by diverse odorant receptors and segregated glomeruli in the main olfactory bulb (MOB) are assembled into integrated cortical representations. Combining patterned optical microstimulation of MOB with in vivo electrophysiological recordings in anterior piriform cortex (PCx), we assessed how cortical neurons decode complex activity patterns distributed across MOB glomeruli. PCx firing was insensitive to single-glomerulus photostimulation. Instead, individual cells reported higher-order combinations of coactive glomeruli resembling odor-evoked sensory maps. Intracellular recordings revealed a corresponding circuit architecture providing each cortical neuron with weak synaptic input from a distinct subpopulation of MOB glomeruli. PCx neurons thus detect specific glomerular ensembles, providing an explicit neural representation of chemical feature combinations that are the hallmark of complex odor stimuli.
Project description:The blue and red shrimp Aristeus antennatus is a demersal marine species harvested by bottom trawling in the Mediterranean Sea, the adjacent Atlantic Ocean (AO) waters, and the Mozambique Channel in the Indian Ocean (IO). As it is considered to be a priority species for sustainable fishing, identification of its genetic stocks and the connectivity between them is essential. Using 12 microsatellite loci we detected at least four genetic stocks distributed in the Western Mediterranean (WM), Eastern Mediterranean (EM), AO, and IO and signals for a possible fifth stock in the Alborán Sea. We detected no additional population structuring within the WM. Thus, although the Almería-Orán Front exerts some isolating effect, high genetic homogeneity and gene flow are present within the WM Basin. The IO stock is genetically closer to the AO stock than to the others; thus, the species dispersion route is more likely via the Atlantic Ocean than via the Red Sea. Large effective population sizes suggest population sustainability, but moderate genetic diversity values indicate to proceed with caution. Our genetic results serve as a basis for species conservation to ensure long-term sustainability of this marine resource.
Project description:Seizures are associated with a reduction in extracellular Ca²(+) concentration ([Ca²(+) ](o) ) and an increase in extracellular K(+) concentration ([K(+) ](o) ). The long-range synchrony observed between distant electrodes during seizures is weak. We hypothesized that changes in extracellular ionic conditions during seizures are sufficient to alter synaptic neuronal responses and synchrony in the neocortex.? We obtained in vivo and in vitro electrophysiologic recordings combined with microstimulation from cat/rat neocortical neurons during seizures and seizure-like ionic conditions. In vitro the [K(+) ](o) was 2.8, 6.25, 8.0, and 12 mm and the [Ca²(+) ](o) was 1.2 and 0.6 mm.During seizures recorded in vivo, we observed abolition of evoked synaptic responses. In vitro, the membrane potential of both regular-spiking and fast-spiking neurons was depolarized in high [K(+) ](o) conditions and hyperpolarized in high [Ca²(+) ](o) conditions. During high [K(+) ](o) conditions, changes in [Ca²(+) ](o) did not affect membrane potential. The synaptic responsiveness of both regular-spiking and fast-spiking neurons was reduced during seizure-like ionic conditions. A reduction in [Ca²(+) ](o) to 0.6 mm increased failure rates but did not abolish responses. However, an increase in [K(+) ](o) to 12 mm abolished postsynaptic responses, which depended on a blockade in axonal spike propagation.We conclude that concomitant changes in [K(+) ](o) and [Ca²(+) ](o) observed during seizures contribute largely to the alterations of synaptic neuronal responses and to the decrease in long-range synchrony during neocortical seizures.