Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging.

Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

Dynamic characteristics of otolith ocular response during counter rotation about dual yaw axes in mice.

The central vestibular system plays an important role in higher neural functions such as self-motion perception and spatial orientation. Its ability to store head angular velocity is called velocity storage mechanism (VSM), which has been thoroughly investigated across a wide range of species. However, little is known about the mouse VSM, because the mouse lacks typical ocular responses such as optokinetic after nystagmus or a dominant time constant of vestibulo-ocular reflex for which the VSM is critical. Experiments were conducted to examine the otolith-driven eye movements related to the VSM and verify its characteristics in mice. We used a novel approach to generate a similar rotating vector as a traditional off-vertical axis rotation (OVAR) but with a larger resultant gravito-inertial force (>1g) by using counter rotation centrifugation. Similar to results previously described in other animals during OVAR, two components of eye movements were induced, i.e. a sinusoidal modulatory eye movement (modulation component) on which a unidirectional nystagmus (bias component) was superimposed. Each response is considered to derive from different mechanisms; modulations arise predominantly through linear vestibulo-ocular reflex, whereas for the bias, the VSM is responsible. Data indicate that the mouse also has a well-developed vestibular system through otoliths inputs, showing its highly conserved nature across mammalian species. On the other hand, to reach a plateau state of bias, a higher frequency rotation or a larger gravito-inertial force was considered to be necessary than other larger animals. Compared with modulation, the bias had a more variable profile, suggesting an inherent complexity of higher-order neural processes in the brain. Our data provide the basis for further study of the central vestibular system in mice, however, the underlying individual variability should be taken into consideration.

Commissural effects in the otolith system.

We examined whether otolith-activated second- and third-order vestibular nucleus neurons received commissural inhibition from the contralateral otolithic macula oriented in the same geometric plane. For this purpose we performed intracellular recording in vestibular nucleus neurons after stimulation of the ipsi- and contralateral utricular and saccular nerves. More than half (41/72) of the utricular-activated second-order vestibular nucleus neurons received commissural inhibition from the contralateral utricular nerve. The remaining neurons (31/72) showed no visible response to contralateral utricular nerve stimulation. About half (17/36) of utricular-activated third-order neurons also received commissural inhibition from the contralateral utricular nerve. Approximately 10% (7/67) of saccular-activated second-order vestibular neurons received polysynaptic commissural inhibition, whereas 16% (11/67) received commissural facilitation. The majority (49/67) of saccular second-order vestibular neurons, and almost all (22/23) third-order neurons, showed no visible response to stimulation of the contralateral saccular nerve. The present findings suggest that many utricular-activated vestibular nucleus neurons receive commissural inhibition, which may provide a mechanism for increasing the sensitivity of vestibular neurons to horizontal linear acceleration and lateral tilt of the head. Commissural inhibition in the saccular system was less prominent than in the utricular system.

Properties of utricular and saccular nerve-activated vestibulocerebellar neurons in cats.

Properties of otolith inputs to vestibulocerebellar neurons were investigated in 14 adult cats. In the vestibular nuclei, we recorded single-unit activities that responded orthodromically after stimulation of the utricular and/or saccular nerves and antidromically after stimulation of the cerebellum (uvula-nodulus and anterior vermis). Descending axonal projections to the spinal cord were also examined by antidromic stimulation of the caudal end of the C1 segment. Forty-seven otolith-activated neurons that projected to the uvula-nodulus were recorded. Thirteen (28%) of the 47 neurons received convergent inputs from the utriculus and sacculus. The remaining 34 (72%) vestibular neurons were non-convergent neurons: 18 (38%) received utricular input alone, and 16 (34%) received saccular input alone. Most (35/47) vestibulocerebellar neurons were located in the descending vestibular nucleus and only one of these projected to the spinal cord. Seven of the 47 vestibulocerebellar neurons were located in the lateral vestibular nucleus and most of these neurons projected to the spinal cord. The remaining neurons were located in group X (two neurons) and the superior vestibular nucleus (three neurons). In a different series of experiments, 37 otolith-activated vestibular neurons were tested to determine whether they projected to the uvula-nodulus and/or the anterior vermis. Nineteen of the 37 neurons projected to the anterior vermis, 13/37 projected to the uvula-nodulus, and 5/37 projected to both. The utricular and/or saccular nerve-activated vestibulocerebellar neurons projected to not only the uvulanodulus, but also to the anterior vermis. In summary, the results of this study showed that vestibular neurons receiving inputs from the utriculus and/or sacculus projected to the cerebellar cortex. This indirect otolith-cerebellar pathway terminated both in the anterior lobe and in the uvula/nodulus.

Convergence patterns of the posterior semicircular canal and utricular inputs in single vestibular neurons in cats.

The convergence of the posterior semicircular canal (PC) and utricular (UT) inputs in single vestibular nuclei neurons was studied intracellularly in decerebrate cats. A total of 160 vestibular neurons were orthodromically activated by selective stimulation of the PC and the UT nerve and classified according to whether or not they were antidromically activated from the spinal cord and oculomotor nuclei into vestibulospinal (VS), vestibulooculospinal (VOS), vestibuloocular (VO), and unidentified vestibular neurons. Fifty-three (33%) of 160 vestibular neurons received convergent inputs from both the PC and UT nerves. Seventy-nine (49%) vestibular neurons responded to PC inputs alone, and 28 (18%) neurons received inputs only from the UT nerve. Of 53 convergent neurons, 8 (15%) were monosynaptically excited from both nerves. Thirty-five (66%) received monosynaptic excitatory inputs from the PC nerve and polysynaptic excitatory or inhibitory inputs from the UT nerve, or vice versa. Approximately one-third of VS and VOS neurons received convergent inputs. A majority of the VS neurons descended to the spinal cord through the lateral vestibulospinal tract, while almost all the VOS neurons descended to the spinal cord through the medial vestibulospinal tract. The convergent neurons were found in all vestibular nuclei but more in the lateral nucleus and descending nucleus. The VS neurons were more numerous than VO neurons or VOS neurons.

Convergence of posterior semicircular canal and saccular inputs in single vestibular nuclei neurons in cats.

Convergence between posterior canal (PC) and saccular (SAC) inputs in single vestibular nuclei neurons was investigated in decerebrated cats. Postsynaptic potentials were recorded intracellularly after selective stimulation of the SAC and PC nerves. Stimulation of either the SAC or PC nerve orthodromically activated 143 vestibular nuclei neurons. Of these, 61 (43%) were antidromically activated by stimulation of the C1-C2 junction, 14 (10%) were antidromically activated by stimulation of the oculomotor or trochlear nucleus, and 14 (10%) were antidromically activated by stimulation of both the oculomotor or trochlear nucleus and the spinal cord. Fifty-four (38%) neurons were not activated by stimulation of either or both. We named these neurons vestibulospinal (VS), vestibulo-ocular (VO), vestibulooculo-spinal (VOS) and vestibular (V) neurons, respectively. Both PC and SAC inputs converged in 47 vestibular nuclei neurons (26 VS, 2 VO, 6 VOS and 13 V neurons). Of these, 19 received monosynaptic excitatory inputs from both nerves. This input pattern was frequently seen in VS neurons. Approximately half of the convergent VS neurons descended to the spinal cord through the lateral vestibulospinal tract. The remaining half and all the convergent VOS neurons descended to the spinal cord through the medial vestibulospinal tract. Most of the convergent neurons were located in the lateral nucleus or descending nucleus.

Sacculo-ocular reflex connectivity in cats.

The otolith system contributes to the vestibulo-ocular reflexes (VOR) when the head moves linearly in the horizontal plane or tilts relative to gravity. The saccules are thought to detect predominantly accelerations along the gravity vector. Otolith-induced vertical eye movements following vertical linear accelerations are attributed to the saccules. However, information on the neural circuits of the sacculo-ocular system is limited, and the effects of saccular inputs on extraocular motoneurons remain unclear. In the present study, synaptic responses to saccular-nerve stimulation were recorded intracellularly from identified motoneurons of all twelve extraocular muscles. Experiments were successfully performed in eleven cats. Individual motoneurons of the twelve extraocular muscles--the bilateral superior recti (SR), inferior recti (IR), superior obliques (SO), inferior obliques (IO), lateral recti (LR), and medial recti (MR) were identified antidromically following bipolar stimulation of their respective nerves. The saccular nerve was selectively stimulated by a pair of tungsten electrodes after removing the utricular nerve and the ampullary nerves of the semicircular canals. Stimulus intensities were determined from the stimulus-response curves of vestibular N1 field potentials in order to avoid current spread. Intracellular recordings were performed from 129 extraocular motoneurons. The majority of the neurons showed no response to saccular-nerve stimulation. In 17 (30%) of 56 extraocular motoneurons related to vertical eye movements (bilateral SR and IR), depolarizing and/or hyperpolarizing postsynaptic potentials (PSPs) were observed in response to saccular-nerve stimulation. The latencies of PSPs ranged from 2.3 to 8.9 ms, indicating that the extraocular motoneurons received neither monosynaptic nor disynaptic inputs from saccular afferents. The majority of the latencies of the depolarization, including depolarization-hyperpolarization, were in the range of 2.3-3.3 ms. Latencies of hyperpolarizations were typically longer than those of depolarizations. Only one contralateral SO motoneuron of 43 recorded oblique extraocular motoneurons (bilateral SO and IO) showed a depolarization-hyperpolarization in response to saccular-nerve stimulation at a latency of 2.5 ms. None of 30 recorded horizontal extraocular motoneurons (bilateral LR and MR) responded to stimulation of the saccular nerve. The neural linkage in the sacculo-ocular system is relatively weak in comparison to the utriculo-ocular and sacculo-collic systems, suggesting that the role of the sacculo-ocular system in stabilizing eye position may be reduced when compared with utriculo-ocular and semi-circular canal-ocular reflexes.

Saccular and utricular inputs to single vestibular neurons in cats.

Saccular and utricular organs are essential for postural stability and gaze control. Although saccular and utricular inputs are known to terminate on vestibular neurons, few previous studies have precisely elucidated the origin of these inputs. We investigated the saccular and utricular inputs to single vestibular neurons in whole vestibular nuclei of decerebrated cats. Postsynaptic potentials were recorded from vestibular neurons after electrical stimulation of the saccular and utricular nerves. Ascending and descending axonal projections were examined by stimulating the oculomotor/trochlear nuclei and the cervical segment of the spinal cord, respectively. After each experiment, locations of recorded neurons were identified. The recorded neurons (140) were classified into vestibulo-spinal (79), vestibulo-oculo-spinal (9), and vestibulo-ocular (3) neurons based on antidromic responses; 49 other vestibular neurons were unidentified. The majority of recorded neurons were mainly located in the lateral vestibular nucleus. Most of the otolith-activated vestibular nuclei neurons seemed to participate in vestibulospinal reflexes. Of the total 140 neurons recorded, approximately one third (51) received saccular and utricular inputs (convergent neurons). The properties of these 51 convergent neurons were further investigated. Most (33/51) received excitatory postsynaptic potentials (EPSPs) after saccular and utricular nerve stimulation. These results implied that most of the convergent neurons in this study additively coded mixed information for vertical and horizontal linear acceleration. Based on the latencies of convergent neurons, we found that an early integration process for vertical and horizontal linear acceleration existed at the second-order level.

Neuronal organization of the utricular macula concerned with innervation of single vestibular neurons in the cat.

We investigated whether cross-striolar inhibition, which may increase sensitivity to linear acceleration, contributed to utricular (UT) afferent innervation of single vestibular neurons (VNs). Excitatory and inhibitory postsynaptic potentials (EPSPs, IPSPs, respectively) were recorded from VNs after focal stimulation of the UT macula (M). From a total of 83 VNs, 25 (30%) neurons received inputs from both sides of the UTM, and the response patterns were opposite, i.e. cross-striolar inhibition was observed. In roughly 2/3 of these neurons, stimulation of the medial side of the UTM evoked EPSPs, while stimulation of the lateral side evoked IPSPs. In the remaining 1/3 neurons, the response patterns were opposite. Thirty-two (39%) of the 83 neurons received the identical pattern of inputs from both sides of the UTM: EPSPs in 26 neurons and IPSPs in six neurons. Twenty-six (31%) of the 83 neurons received inputs from either the medial or the lateral side of the UTM. These findings suggest that cross-striolar inhibition existed in the UT system, although it was not a dominant circuit that increased the sensitivity as in the saccular system [15].

Canal and otolith inputs to single vestibular neurons in cats.

Convergence of both afferents from the PC and saccular macula, and those from the PC and utricular macula on single vestibular neurons was noted by use of intercellular recording from vestibular neurons. Vestibular neurons were classified VO neurons (vestibulo-ocular proper neurons), VOS (Vestibulo-oculo-spinal neurons sending axon collaterals both to the extraocular motoneuron pools and to the spinal cord), VS neurons (vestibulospinal proper neurons) and V neurons (vestibular neurons without axons to the oculomotor nuclei or the spinal cord) on the basis of whether or not they responded antidromically to stimulation of the oculomotor nuclei and the spinal cord. Of the total 143 vestibular neurons recorded in the series of experiments on convergence of the PC and saccular afferents, 47 neurons (33%) were received inputs from both the PC and saccular nerves. Twenty-six of the 47 convergent neurons were identified as having the nature of VS neurons. Half (13/26) of those were activated monosynaptically from both the PC and saccular nerves. Only one saccular-activated neuron without PC inputs sent an axon to the oculomotor nuclei. In the other series of experiments on the convergence of the PC and utricular afferents, 41 (37%) of 111 vestibular neurons were proved to converge on inputs from both nerves. The majority (35/41) of the neurons received monosynaptic inputs from the PC nerve and polysynaptic EPSP-IPSP sequences from the utricular nerve, or vice versa. The ratio of PC-otolith convergent neurons among utricular-activated neurons (41/54, 76%) was higher than that among saccular activated neurons (47/88, 53%). The percentage of utricular alone neurons without PC inputs (13/111, 12%) was less than that of the saccular alone without PC inputs (41/145, 28%). In conclusion, the convergence of canal and otolith inputs likely contribute mainly to vestibulospinal reflexes including the vestibulocollic reflex, by sending inputs to the neck and other muscles during head inclination which creates the combined stimuli of angular and linear acceleration.

Saccular and utricular inputs to sternocleidomastoid motoneurons of decerebrate cats.

Connections from the otolithic organs to sternocleidomastoid (SCM) motoneurons were studied in 20 decerebrate cats. The electrical stimulation was selective for the saccular or the utricular nerves. Postsynaptic potentials were recorded from antidromically identified SCM motoneurons; these muscles participate mainly in neck rotation and flexion. Partial transections of the brainstem at the level of the obex were performed to identify the possible pathway from the otolithic organs to the SCM motoneurons. Saccular or utricular nerve stimulation mainly evoked inhibitory postsynaptic potentials (IPSPs) in the ipsilateral SCM motoneurons. Some of the sacculus-induced IPSPs were preceded by small-amplitude excitatory PSPs (EPSPs). The latencies of the PSPs ranged from 1.8 to 3.1 ms after saccular nerve stimulation and from 1.7 to 2.8 ms after utricular nerve stimulation, indicating that most of the ipsilateral connections were disynaptic. In the contralateral SCM motoneurons, saccular nerve stimulation had no or faint effects, whereas utricular nerve stimulation evoked EPSPs in about two-thirds of neurons, and no visible PSPs in about one-third of neurons. The latencies of the EPSPs ranged from 1.5 to 2.0 ms, indicating the disynaptic connection. Thus, the results suggest a difference between the two otolithic innervating patterns of SCM motoneurons. After transection of the medial vestibulospinal tract (MVST), saccular nerve stimulation did not evoke IPSPs at all in ipsilateral SCM motoneurons, but some (11/40) neurons showed small-amplitude EPSPs. Most (24/33) of the utricular-activated IPSPs disappeared after transection, whereas the other 9 neurons still indicated IPSPs. In the contralateral SCM motoneurons, no utricular-activated EPSPs were recorded after transection. These MVST transection results suggest that most of the otolith-SCM pathways are located in the MVST at the obex level. However, the results also suggest the possibility that other otolith-SCM pathways exist at the obex level.

Cross-striolar and commissural inhibition in the otolith system.

Neural connections from the saccular and utricular nerves to the ipsilateral vestibular neurons and the commissural effects were studied by using intracellular recordings of excitatory (E) and inhibitory (I) postsynaptic potentials (PSPs) in vestibular neurons of cats after focal stimulation of the saccular and the utricular maculae. Neural circuits from the maculae to vestibular neurons, termed cross-striolar inhibition, may provide a mechanism for increasing the sensitivity to linear acceleration and tilt of the head. It was examined whether secondary vestibular neurons activated by an ipsilateral otolith organ received a commissural inhibition from a contralateral otolith organ that occupied the same geometric plane. Results suggest that utricular-activated vestibular neurons receiving commissural inhibition may provide a mechanism for increasing the sensitivity to horizontal linear acceleration and tilt of the head. The commissural inhibition of the saccular system was much weaker than that of the utricular system.