Cochlear Nucleus Of Guinea Pig Research Articles

Nitric oxide increases gain in the ventral cochlear nucleus of guinea pigs with tinnitus.

Previous work has led to the hypothesis that, during the production of noise-induced tinnitus, higher levels of nitric oxide (NO), in the ventral cochlear nucleus (VCN), increase the gain applied to a reduced input from the cochlea. To test this hypothesis, we noise-exposed 26 guinea pigs, identified evidence of tinnitus in 12 of them and then compared the effects of an iontophoretically applied NO donor or production inhibitor on VCN single unit activity. We confirmed that the mean driven firing rate for the tinnitus and control groups was the same while it had fallen in the non-tinnitus group. By contrast, the mean spontaneous rate had increased for the tinnitus group relative to the control group, while it remained the same for the non-tinnitus group. A greater proportion of units responded to exogenously applied NO in the tinnitus (56%) and non-tinnitus groups (71%) than a control population (24%). In the tinnitus group, endogenous NO facilitated the driven firing rate in 37% (7/19) of neurons and appeared to bring the mean driven rate back up to control levels by a mechanism involving N-methyl-D-aspartic acid (NMDA) receptors. By contrast, in the non-tinnitus group, endogenous NO only facilitated the driven firing rate in 5% (1/22) of neurons and there was no facilitation of driven rate in the control group. The effects of endogenous NO on spontaneous activity were unclear. These results suggest that NO is involved in increasing the gain applied to driven activity, but other factors are also involved in the increase in spontaneous activity.

Cholinergic responses of acoustically-characterized cochlear nucleus neurons: An in vivo iontophoretic study in Guinea pig

The responses of guinea pig cochlear nucleus neurons to in vivo iontophoretic application of various neurotransmitter agonists were recorded with extracellular multi-barrelled electrodes. Where possible, neurons were physiologically identified using strict criteria. Emphasis was placed on the action of cholinergic agonists in relation to the possible action of olivocochlear collateral innervation. Excitatory responses (increase in action potential firing) to glutamate were confirmed in a number of neuronal response types. Application of acetylcholine (ACh) or the broad spectrum cholinergic agonist carbachol produced reliable excitatory responses in about 47% of neurons (n = 29 out of 61 neurons). The remaining neurons were unresponsive to cholinergic agonists and no inhibitory responses were observed. Cholinergic responses were more common in dorsal cochlear nucleus (DCN) (73% of 30 neurons tested) than in ventral cochlear nucleus (VCN) (23% of 31 neurons). Of the total neuron sample in which cholinergic responses were investigated, 41 neurons were able to be categorized according to established acoustic response features. Excitatory responses to cholinergic agonists were seen in “Pauser-buildup” (Pb) and “Transient chopper” (Ct) response types. Primary-like neurons (PL and Pn) as well as “Onset chopper” (Oc) neurons (n = 6) were unresponsive to either ACh or carbachol. Oc neurons also did not show any effect on their acoustic responses. Robust cholinergic responses were also seen in several VCN and DCN neurons that were either unresponsive to sound, or had acoustic response properties that did not fit standard classification. The results suggest a relatively more robust cholinergic innervation of DCN compared to VCN. The excitatory cholinergic responses of some Ct neurons and the lack of effect on Oc neurons are consistent with previous results in mouse brain slice studies, but are in conflict with reports of medial olivocochlear collateral excitatory responses in onset-type neurons in vivo. The results also indicate that a number of neurons of unknown identity may also receive cholinergic input.

Dual Coding of Frequency Modulation in the Ventral Cochlear Nucleus.

Frequency modulation (FM) is a common acoustic feature of natural sounds and is known to play a role in robust sound source recognition. Auditory neurons show precise stimulus-synchronized discharge patterns that may be used for the representation of low-rate FM. However, it remains unclear whether this representation is based on synchronization to slow temporal envelope (ENV) cues resulting from cochlear filtering or phase locking to faster temporal fine structure (TFS) cues. To investigate the plausibility of those encoding schemes, single units of the ventral cochlear nucleus of guinea pigs of either sex were recorded in response to sine FM tones centered at the unit's best frequency (BF). The results show that, in contrast to high-BF units, for modulation depths within the receptive field, low-BF units (<4 kHz) demonstrate good phase locking to TFS. For modulation depths extending beyond the receptive field, the discharge patterns follow the ENV and fluctuate at the modulation rate. The receptive field proved to be a good predictor of the ENV responses for most primary-like and chopper units. The current in vivo data also reveal a high level of diversity in responses across unit types. TFS cues are mainly conveyed by low-frequency and primary-like units and ENV cues by chopper and onset units. The diversity of responses exhibited by cochlear nucleus neurons provides a neural basis for a dual-coding scheme of FM in the brainstem based on both ENV and TFS cues.SIGNIFICANCE STATEMENT Natural sounds, including speech, convey informative temporal modulations in frequency. Understanding how the auditory system represents those frequency modulations (FM) has important implications as robust sound source recognition depends crucially on the reception of low-rate FM cues. Here, we recorded 115 single-unit responses from the ventral cochlear nucleus in response to FM and provide the first physiological evidence of a dual-coding mechanism of FM via synchronization to temporal envelope cues and phase locking to temporal fine structure cues. We also demonstrate a diversity of neural responses with different coding specializations. These results support the dual-coding scheme proposed by psychophysicists to account for FM sensitivity in humans and provide new insights on how this might be implemented in the early stages of the auditory pathway.

Auditory-somatosensory bimodal stimulation desynchronizes brain circuitry to reduce tinnitus in guinea pigs and humans.

The dorsal cochlear nucleus is the first site of multisensory convergence in mammalian auditory pathways. Principal output neurons, the fusiform cells, integrate auditory nerve inputs from the cochlea with somatosensory inputs from the head and neck. In previous work, we developed a guinea pig model of tinnitus induced by noise exposure and showed that the fusiform cells in these animals exhibited increased spontaneous activity and cross-unit synchrony, which are physiological correlates of tinnitus. We delivered repeated bimodal auditory-somatosensory stimulation to the dorsal cochlear nucleus of guinea pigs with tinnitus, choosing a stimulus interval known to induce long-term depression (LTD). Twenty minutes per day of LTD-inducing bimodal (but not unimodal) stimulation reduced physiological and behavioral evidence of tinnitus in the guinea pigs after 25 days. Next, we applied the same bimodal treatment to 20 human subjects with tinnitus using a double-blinded, sham-controlled, crossover study. Twenty-eight days of LTD-inducing bimodal stimulation reduced tinnitus loudness and intrusiveness. Unimodal auditory stimulation did not deliver either benefit. Bimodal auditory-somatosensory stimulation that induces LTD in the dorsal cochlear nucleus may hold promise for suppressing chronic tinnitus, which reduces quality of life for millions of tinnitus sufferers worldwide.

Effects of Cochlear Trauma on BDNF Expression in Guinea Pig Cochlear Nucleus and Inferior Colliculus

Hearing loss caused by cochlear damage results in a variety of plastic changes in the central auditory pathways. One of these is hyperactivity, i.e. increased spontaneous firing rates, which may be involved in the generation of tinnitus, a phantom auditory sensation. The mechanism behind this synaptic plasticity is still uncertain but there may be a role for Brain Derived Neurotrophic Factor (BDNF). The expression of BDNF is activity dependent and BDNF can modulate synaptic plasticity leading to changes in excitability. In the present study we investigated the effects of two different types of cochlear trauma, mechanical and acoustic, at two different time points after trauma, on 1) peripheral hearing loss, 2) hyperactivity in inferior Colliculus (IC) and 3) BDNF protein expression in Cochlear Nucleus (CN) and IC of guinea pigs. BDNF protein expression was determined using ELISA. Although there was no significant difference in the amount of hearing loss between acoustic trauma and mechanical trauma animals, single neuron recordings showed higher levels of hyperactivity after mechanical trauma than after acoustic trauma at two weeks post-recovery from cochlear trauma.In addition, results showed an increase of BDNF levels in the ipsilateral CN and contralateral IC at 2 weeks after mechanical but not after acoustic trauma. BDNF levels recovered to sham control levels in both structures at 6 weeks after cochlear trauma even though hyperactivity remained higher compared to sham surgery animals at the same timepoint. The results suggest a possible time dependent role for BDNF in modulating synaptic plasticity and excitability after mechanical trauma to the cochlea.

Multiple origins of cholinergic innervation of the cochlear nucleus

Acetylcholine (Ach) affects a variety of cell types in the cochlear nucleus (CN) and is likely to play a role in numerous functions. Previous work in rats suggested that the acetylcholine arises from cells in the superior olivary complex, including cells that have axonal branches that innervate both the CN and the cochlea (i.e. olivocochlear cells) as well as cells that innervate only the CN. We combined retrograde tracing with immunohistochemistry for choline acetyltransferase to identify the source of ACh in the CN of guinea pigs. The results confirm a projection from cholinergic cells in the superior olivary complex to the CN. In addition, we identified a substantial number of cholinergic cells in the pedunculopontine tegmental nucleus (PPT) and the laterodorsal tegmental nucleus (LDT) that project to the CN. On average, the PPT and LDT together contained about 26% of the cholinergic cells that project to CN, whereas the superior olivary complex contained about 74%. A small number of additional cholinergic cells were located in other areas, including the parabrachial nuclei.The results highlight a substantial cholinergic projection from the pontomesencephalic tegmentum (PPT and LDT) in addition to a larger projection from the superior olivary complex. These different sources of cholinergic projections to the CN are likely to serve different functions. Projections from the superior olivary complex are likely to serve a feedback role, and may be closely tied to olivocochlear functions. Projections from the pontomesencephalic tegmentum may play a role in such things as arousal and sensory gating. Projections from each of these areas, and perhaps even the smaller sources of cholinergic inputs, may be important in conditions such as tinnitus as well as in normal acoustic processing.

A 3-D 160-Site Microelectrode Array for Cochlear Nucleus Mapping

A 3-D application-specific microelectrode array has been developed for physiological studies in guinea pig cochlear nucleus (CN). The batch-fabricated silicon probes contain integrated parylene cables and use a boron etch-stop to define 15μm-thick shanks and limit tissue displacement. Targeting the ventral (three probes) and dorsal (two probes) subnuclei, the custom four-shank 32-site probes are combined in a slotted block platform having a 1.18-mm (2) footprint. The device has permitted, for the first time, high-density 3-D in vivo studies of ventral CN to dorsal CN connections, stimulating with 1000 μm (2) sites in one subnucleus while recording with 177 μm (2) sites in the other. Through these experiments, it has demonstrated the efficacy of bimodal silicon arrays to better understand the central nervous system at the circuit level. The 160 electrode sites also provide a high-density neural interface, which is an essential aspect of auditory prosthesis prototypes.

Time course of neuronal and synaptic plasticity in dorsal cochlear nucleus of guinea pig following chronic kanamycin-induced deafness

We investigated the time course of the plasticity in fusiform cell (FC) and at auditory nerve (AN) synapse on FC (AN/FC synapse) following chronic kanamycin-induced deafness. Guinea pigs were treated with kanamycin sulfate by subcutaneous injection at dose of 500mg/kg/day for 7days. Ultrastructural changes in FC and AN/FC synapse were observed, and local insulin-like growth factor 1 (IGF-1) mRNA was quantified using quantitative real time PCR at 1, 7, 14, 28, 70 and 140days after kanamycin treatment. The average threshold was 46.46±3.45, 80.63±5.95 and 103.95±6.59dB SPL respectively at 1, 7 and 14days, and the threshold was statistically unchanged at 28, 70 and 140days in comparison with the 14day group. Mitochondrial swelling in FC and at AN/FC synapse was progressive at 7, 14 and 28days. Moreover, the thickness of the postsynaptic densities increased at 1, 7 and 14days. Finally, there was a persistent upregulation in local IGF-1 mRNA at 7, 14, 28 and 70days. These changes in the ultrastructure of AN/FC synapse and FC, and upregulation of local IGF-1mRNA were no longer present at 140days. Our results indicate that the effects of kanamycin on the ultrastructure of FC and AN/FC synapse are progressive. However, FC and AN/FC synapse are capable of reviving and remodeling after kanamycin-induced lesion and incomplete deafferentation. Additionally, local IGF-1 might play a role in the lesion- and deafness-induced plasticity in FC and at AN/FC synapse following chronic kanamycin-induced deafness.

Tonotopic responses in the inferior colliculus following electrical stimulation of the dorsal cochlear nucleus of guinea pigs

Tonotopic responses in the inferior colliculus following electrical stimulation of the dorsal cochlear nucleus of guinea pigs

Electrically Evoked Responses in Onset Chopper Neurons in Guinea Pig Cochlear Nucleus

Extracellular recordings were obtained from single cochlear nucleus neurons in guinea pigs anesthetized with Nembutal and Hypnorm. Neurons were classified by their spontaneous firing rates and responses to acoustic stimuli. In addition, electrical shocks were applied to the midline at the level of the IVth ventricle and spike responses were recorded. Spikes were evoked by shocks only in neurons that were classified as onset choppers (O(c)). The shock-evoked spikes could be extinguished by acoustically evoked action potentials in the same neurons. In roughly 30% of the sample of O(c) neurons, quantitative aspects of the timing of this extinction were not compatible with the shock-evoked spike being antidromically conducted from O(c) output axons. Together with the presence of temporal jitter at high shock rates, the data suggest the possibility that at least some of the shock-evoked spikes may be generated by excitatory synaptic input to the O(c) neurons, most likely from the collaterals of the medial olivocochlear system (MOCS), whose axons pass close to the floor of the IVth ventricle. This excitatory synaptic input may operate to modulate the activity of O(c) neurons in addition to MOCS actions in the auditory periphery.

Cells in Auditory Cortex that Project to the Cochlear Nucleus in Guinea Pigs

Fluorescent retrograde tracers were used to identify the cells in auditory cortex that project directly to the cochlear nucleus (CN). Following injection of a tracer into the CN, cells were labeled bilaterally in primary auditory cortex and the dorsocaudal auditory field as well as several surrounding fields. On both sides, the cells were limited to layer V. The size of labeled cell bodies varied considerably, suggesting that different cell types may project to the CN. Cells ranging from small to medium in size were present bilaterally, whereas the largest cells were labeled only ipsilaterally. In optimal cases, the extent of dendritic labeling was sufficient to identify the morphologic class. Many cells had an apical dendrite that could be traced to a terminal tuft in layer I. Such "tufted" pyramidal cells were identified both ipsilateral and contralateral to the injected CN. The results suggest that the direct pathway from auditory cortex to the cochlear nucleus is substantial and is likely to play a role in modulating the way the cochlear nucleus processes acoustic stimuli.

Pathways from auditory cortex to the cochlear nucleus in guinea pigs

The inferior colliculus (IC) and superior olivary complex (SOC) are important sources of descending pathways to the cochlear nucleus. The IC and SOC are also targets of direct projections from the auditory cortex but it is not known if cortical axons contact the cells that project to the cochlear nucleus. Multi-labeling techniques were used to address this question in guinea pigs. A fluorescent anterograde tracer was injected into temporal cortex to label corticofugal axons. Different fluorescent tracers were injected into one or both cochlear nuclei to label olivary and collicular cells. The brain was subsequently processed for fluorescence microscopy and the IC and SOC were examined for apparent contacts between cortical axons and retrogradely labeled cells. The results suggest that cortical axons contact cochlear nucleus-projecting cells in both IC and SOC. In both regions, contacts were more numerous on the side ipsilateral to the injected cortex. In the IC, the contacted cells projected ipsilaterally or contralaterally to the CN. In the SOC, the contacted cells projected ipsilaterally, contralaterally or bilaterally to the CN. We conclude that auditory cortex is in a position to modulate descending pathways from both the IC and SOC to the cochlear nucleus.

Projections from the trigeminal nuclear complex to the cochlear nuclei: A retrograde and anterograde tracing study in the guinea pig

In addition to input from auditory centers, the cochlear nucleus (CN) receives inputs from nonauditory centers, including the trigeminal sensory complex. The detailed anatomy, however, and the functional implications of the nonauditory innervation of the auditory system are not fully understood. We demonstrated previously that the trigeminal ganglion projects to CN, with terminal labeling most dense in the marginal cell area and secondarily in the magnocellular area of the ventral cochlear nucleus (VCN). We continue this line of study by investigating the projection from the spinal trigeminal nucleus to CN in guinea pig. After injections of the retrograde tracers FluoroGold or biotinylated dextran amine (BDA) in VCN, labeled cells were found in the spinal trigeminal nuclei, most densely in the pars interpolaris and pars caudalis with ipsilateral dominance. The anterograde tracers Fluoro-Ruby or BDA were stereotaxically injected into the spinal trigeminal nucleus. Most labeled puncta were found in the marginal area of VCN and the fusiform cell layer of dorsal cochlear nucleus (DCN). A smaller number of labeled puncta was located in the molecular and deep layers of DCN and the magnocellular area of VCN. The trigeminal projection to CN may provide somatosensory information necessary for pursuing a sound source or for vocal production. These projections may have a role in the generation and modulation of tinnitus.

Auditory cortical projections to the cochlear nucleus in guinea pigs

We used anterograde tracing techniques to examine projections from auditory cortex to the cochlear nucleus in guinea pigs. Following injection of dextrans into the temporal cortex, labeled axons were present bilaterally in the cochlear nucleus. The distribution of boutons within the cochlear nucleus was similar on the two sides. The majority of boutons was usually located on the ipsilateral side. Most of the boutons were located in the granule cell areas, where many small boutons and a few larger, mossy-type endings were labeled. Additional small, labeled boutons were found in all layers of the dorsal cochlear nucleus, with the majority located in the fusiform cell layer. Labeled boutons were also present in the ventral cochlear nucleus, where they were located in the small cell cap as well as magnocellular parts of both posteroventral and anteroventral cochlear nucleus. Similar results were obtained with injections restricted to primary auditory cortex or to the dorsocaudal auditory field. The results illustrate direct cortical projections to the cochlear nucleus that are likely to modulate the activity in a number of ascending auditory pathways.

An immunogold investigation of the distribution of GABA and glycine in nerve terminals on the somata of spherical bushy cells in the anteroventral cochlear nucleus of guinea pig.

Spherical bushy neurons in the anteroventral cochlear nucleus receive glutamatergic primary terminals from the cochlear nerve and terminals of noncochlear (i.e. nonprimary) origin, many of which colocalize gamma-aminobutyric acid (GABA) and glycine. Here the relationship between GABA and glycine in these terminals has been investigated using postembedding immunogold labelling. A significant negative correlation was found between the density of terminal labelling for GABA and for glycine in four guinea pigs. Terminals could be divided into three categories, GABA-only, glycine-only, or colocalizing depending on whether they had a significantly higher labelling density for either amino acid than the primary terminals. The overall labelling density in all four animals was significantly greater for GABA in GABA-only terminals than colocalizing ones but similar for glycine in both. Within the terminals, the labelling density over synaptic vesicles, nonvesicular regions of cytoplasm and mitochondria was also investigated. No significant difference was detected in the labelling density of vesicles compared with nonvesicular regions for either amino acid. However, a significant difference was found between the overall labelling density over mitochondria and nonvesicular regions for both. There was also significantly more mitochondrial GABA labelling in GABA-only terminals compared to colocalizing terminals but mitochondrial glycine labelling was similar in glycine-only and colocalizing terminals. Thus the level of GABA is higher in single than in colocalizing terminals, particularly in the mitochondria, but similar for glycine in both. It is possible therefore that the presence of glycine affects the level of GABA in the nonprimary terminals but that the presence of GABA does not affect the level of glycine.

The projection from auditory cortex to cochlear nucleus in guinea pigs: an in vivo anatomical and in vitro electrophysiological study.

Previous anatomical experiments have demonstrated the existence of a direct, bilateral projection from the auditory cortex (AC) to the cochlear nucleus (CN). However, the precise relationship between the origin of the projection in the AC and the distribution of axon terminals in the CN is not known. Moreover, the influence of this projection on CN principal cells has not been studied before. The aim of the present study was two-fold. First, to extend the anatomical data by tracing anterogradely the distribution of cortical axons in the CN by means of restricted injections of biotinylated dextran amine (BDA) in physiologically characterized sites in the AC. Second, in an in vitro isolated whole brain preparation (IWB), to assess the effect of electrical stimulation of the AC on CN principal cells from which intracellular recordings were derived. BDA injections in the tonotopically organized primary auditory cortex and dorsocaudal auditory field at high and low best frequency (BF) sites resulted in a consistent axonal labeling in the ipsilateral CN of all injected animals. In addition, fewer labeled terminals were observed in the contralateral CN, but only in the animals subjected to injections in low BF region. The axon terminal fields consisting of boutons en passant or terminaux were found in the superficial granule cell layer and, to a smaller extent, in the three CN subdivisions. No axonal labeling was seen in the CN as result of BDA injection in the secondary auditory area (dorsocaudal belt). In the IWB, the effects of ipsilateral AC stimulation were tested in a population of 52 intracellulary recorded and stained CN principal neurons, distributed in the three CN subdivisions. Stimulation of the AC evoked slow late excitatory postsynaptic potentials (EPSPs) in only two cells located in the dorsal CN. The EPSPs were induced in a giant and a pyramidal cell at latencies of 20 ms and 33 ms, respectively, suggesting involvement of polysynaptic circuits. These findings are consistent with anatomical data showing sparse projections from the AC to the CN and indicate a limited modulatory action of the AC on CN principal cells.

TrkB levels in the cochlear nucleus after unilateral cochlear ablation: correlations with post-lesion plasticity

Tyrosine kinase B (TrkB) levels in the adult guinea pig cochlear nucleus (CN) were determined from Western blots for up to 60 days after unilateral cochlear ablation (UCA). Compared to TrkB levels on the intact side, those on the lesioned side were elevated in the anteroventral CN (AVCN) at 7 and 60 days and in the posteroventral CN (PVCN) at 30 days. TrkB levels were depressed in the AVCN and the dorsal CN (DCN) at 30 days. Elevations in the AVCN on the lesioned side at 7 days coincided with a period of synaptogenesis. Other changes were temporally related to up- or downregulations of transmitter release and synaptic receptor activities that were evident after UCA. The findings suggest that changes in signaling through TrkB may contribute to the plasticities in the CN that were evident after UCA.

Origins of projections from the inferior colliculus to the cochlear nucleus in guinea pigs.

We used retrograde tracing techniques to examine the projections from the inferior colliculus to the cochlear nucleus in guinea pigs. Following injection of a retrograde tracer into one cochlear nucleus, labeled cells were found bilaterally in all subdivisions of the inferior colliculus. The majority of cells were located in the central nucleus and external cortex; relatively few cells were located in the dorsal cortex. Multipolar (stellate) cells were labeled in all subdivisions of the inferior colliculus. In the central nucleus, disk-shaped cells were also labeled. To determine whether individual collicular neurons send collateral projections to the cochlear nuclei on both sides, we injected different fluorescent tracers into left and right cochlear nuclei in the same animal. The inferior colliculi contained very few double-labeled cells, indicating that the projections to ipsilateral and contralateral cochlear nuclei originate from separate populations of cells.

Multi-channel spike detection and sorting using an array processing technique

In many brain regions, high levels of background activity can obscure action potentials of neurons close to a recording electrode, particularly in the presence of a stimulus. As recorded on a multi-channel electrode array, this neural noise appears to be highly correlated among the channels. We have developed an array processing technique to remove the correlated component of noise, improving the signal-to-noise ratio. In addition, we show that the weighting vector of the array algorithm can be manipulated to facilitate sorting of the action potentials. An example of the technique applied to the guinea pig cochlear nucleus is shown.

Histological and physiological effects of the central auditory prosthesis: surface versus penetrating electrodes

To rehabilitate profoundly deaf patients who are not suitable for cochlear implants, central auditory prostheses have been implanted. To compare two possible electrode configurations - penetrating and surface ones - electrical stimulation of the cochlear nucleus with both types of arrays was tested on guinea pigs and cats. Electrophysiological, autoradiographic and histological measures were used to study effects of the central auditory prostheses on the auditory pathway. The results showed that a successful electrically evoked auditory brainstem response could be recorded with both surface and penetrating electrodes in cats and guinea pigs. In guinea pigs the penetrating electrodes had advantages over surface arrays in the sense of lower thresholds and wider dynamic ranges. In cats penetrating electrodes showed lower thresholds than surface ones. In cats and guinea pigs stimulated with either surface or penetrating electrodes, evoked 2-deoxyglucose (2-DG) label was found in the auditory pathway from the cochlear nucleus to the inferior colliculus. No non-auditory tissues were found with evoked 2-DG label. Histological results showed that in subdivisions of the guinea pig cochlear nucleus stimulated with penetrating electrodes the neurone density was decreased, and the mean soma area was increased compared with the control side. In the cat, penetrating electrodes were associated only with increased mean soma area in parts of the stimulated cochlear nucleus. These results suggest that the physiological advantages of penetrating electrodes over surface ones were achieved with some trade-off in safety, especially in the guinea pig.