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The effects of DSP-4 administration on the dopamine beta hydroxylase-immunoreactivity (DBH-ir) in the inferior colliculus (IC). A schematic representation of DBH-ir in the IC from the caudal to the rostral part. (A) Drawings of the DBH-ir in control animals. (B) Drawings of the DBH-ir in DSP-4-treated animals. Notice how the immunoreactivity almost disappears in treated animals. (C) The mean gray value of the distribution of DBH-ir in control animals at different rostrocaudal levels and subdivisions. (D) The mean gray value of the distribution of DBH-ir in DSP-4-treated animals at different rostrocaudal levels and subdivisions. (E) The percent reduction in DBH-ir following DSP-4 treatment. The interaural level for the coronal placement is displayed in millimeters. The bars represent means ± SD of 24 stained sections. See the provided list for abbreviations.
Source publication
The inferior colliculus (IC) and the locus coeruleus (LC) are two midbrain nuclei that integrate multimodal information and play a major role in novelty detection to elicit an orienting response. Despite the reciprocal connections between these two structures, the projection pattern and target areas of the LC within the subdivisions of the rat IC a...
Citations
... Indeed, a number of studies show that processing and FIGURE 2 | Summary of inferior colliculus afferent and efferent connections. Afferents: Ascending (later lemniscus): cochlear nuclei (Klepper and Herbert, 1991;Cant and Benson, 2006), superior olive (Cant and Benson, 2006), lateral lemniscus (Zhang et al., 1998;Saalmann et al., 2006;Kelly et al., 2009;Chen et al., 2018); Ascending (other): substantia nigra/VTA (Takada et al., 1987;Kemel et al., 1988;Yasui et al., 1991;Deniau and Chevalier, 1992;Moriizumi et al., 1992), locus coeruleus (Klepper and Herbert, 1991;Hormigo et al., 2012), dorsal raphe (Hurley and Pollak, 2001;Hurley, 2006;Hurley and Sullivan, 2012), pedunculopontine nucleus (Noftz et al., 2020), periaqueductal gray (Dujardin and Jürgens, 2005), cerebellum (Huffman and Henson, 1990); Super Colliculus (Adams, 1980;Clerici and Coleman, 1987;Stepniewska et al., 2000;Nodal et al., 2005); Descending: medial geniculate (Driscoll and Tadi, 2020), other thalamic structures (Kuwabara and Zook, 2000), amygdala (Hopkins and Holstege, 1978;Marsh et al., 2002), auditory cortex (Games and Winer, 1988); visual, motor, somatosensory cortices (Cooper and Young, 1976). Efferents: Ascending (inferior brachium): medial geniculate (Oliver, 1984b;Oliver et al., 1991;Peruzzi et al., 1997;Smith et al., 2006); Ascending (other): other thalamic nuclei (Kudo and Niimi, 1980;Senatorov and Hu, 2002;Smith et al., 2006), auditory cortex (Schofield et al., 2011;Xiong et al., 2015;Schofield and Beebe, 2019;Lesicko et al., 2020); Super Colliculus (Van Buskirk, 1983); Descending: auditory brainstem (Huffman and Henson, 1990), periaqueductal gray (Santos et al., 2003;Reimer et al., 2008). ...
Post-mortem neuropathological and in vivo neuroimaging methods have demonstrated the vulnerability of the inferior colliculus to the sequelae of thiamine deficiency as occurs in Wernicke-Korsakoff Syndrome (WKS). A rich literature in animal models ranging from mice to monkeys—including our neuroimaging studies in rats—has shown involvement of the inferior colliculi in the neural response to thiamine depletion, frequently accomplished with pyrithiamine, an inhibitor of thiamine metabolism. In uncomplicated alcoholism (i.e., absent diagnosable neurological concomitants), the literature citing involvement of the inferior colliculus is scarce, has nearly all been accomplished in preclinical models, and is predominately discussed in the context of ethanol withdrawal. Our recent work using novel, voxel-based analysis of structural Magnetic Resonance Imaging (MRI) has demonstrated significant, persistent shrinkage of the inferior colliculus using acute and chronic ethanol exposure paradigms in two strains of rats. We speculate that these consistent findings should be considered from the perspective of the inferior colliculi having a relatively high CNS metabolic rate. As such, they are especially vulnerable to hypoxic injury and may be provide a common anatomical link among a variety of disparate insults. An argument will be made that the inferior colliculi have functions, possibly related to auditory gating, necessary for awareness of the external environment. Multimodal imaging including diffusion methods to provide more accurate in vivo visualization and quantification of the inferior colliculi may clarify the roles of brain stem nuclei such as the inferior colliculi in alcoholism and other neuropathologies marked by altered metabolism.
... The IC may receive motor-related signals247 via the descending projections from the motor cortex (Olthof et al., 2019). Cholinergic inputs 248 from the peduncular pontine nucleus (Farley et al., 1983; Motts and Schofield, 2009), which249is part of the midbrain locomotion region (Lee et al., 2014; Caggiano et al., 2018), and 250 noradrenergic inputs from the locus coeruleus (Klepper and Herbert, 1991;Hormigo et al., ...
The inferior colliculus (IC) is the major midbrain auditory integration center, where virtually all ascending auditory inputs converge. Although the IC has been extensively studied for sound processing, little is known about the neural activity of the IC in moving subjects, as frequently happens in natural hearing conditions. Here, by recording neural activity in walking mice, we show that the activity of IC neurons is strongly modulated by locomotion, even in the absence of sound stimuli. Similar modulation was also found in hearing-impaired mice, demonstrating that IC neurons receive non-auditory, locomotion-related neural signals. Sound-evoked activity was attenuated during locomotion, and this attenuation increased frequency selectivity across the neuronal population, while maintaining preferred frequencies. Our results suggest that during behavior, integrating movement-related and auditory information is an essential aspect of sound processing in the IC.
... Second, the IC receives inputs from midbrain cholinergic neurons in the peduncular pontine nucleus (Farley et al., 1983;Motts and Schofield, 2009), which is part of the midbrain locomotion region (Lee et al., 2014;Caggiano et al., 2018). Third, the IC receives noradrenergic inputs from the locus coeruleus (Klepper and Herbert, 1991;Hormigo et al., 2012), which are likely to be active during locomotion (Reimer et al., 2016). Therefore, IC neurons could receive rich information about body movement via both efference copy like signals from motor-related regions and somatosensory feedback. ...
The inferior colliculus (IC) is the major midbrain auditory integration center, where virtually all ascending auditory inputs converge. Although the IC has been extensively studied for sound processing, little is known about the neural activity of the IC in moving subjects, as frequently happens in natural hearing conditions. Here we show, by recording the IC neural activity in walking mice, the activity of IC neurons is strongly modulated by locomotion in the absence of sound stimulus presentation. Similar modulation was also found in deafened mice, demonstrating that IC neurons receive non-auditory, locomotion-related neural signals. Sound-evoked activity was attenuated during locomotion, and the attenuation increased frequency selectivity across the population, while maintaining preferred frequencies. Our results suggest that during behavior, integrating movement-related and auditory information is an essential aspect of sound processing in the IC.
... To begin to examine contributions of NE from LC neurons we employed the DSP-4 neurotoxin, a small-molecule toxin that, through ill-defined mechanisms, targets the noradrenergic transporter and selectively destroys terminals originating from LC, and in some cases cell bodies in the LC. 38 To determine an optimal treatment strategy for reducing NE cell bodies in the LC, groups of C57BL/6 mice received either one injection of saline, or one (50 or 75 mg/kg) or two (50 mg/kg; 7 days apart) injections of DSP-4. One week after the last injection, mice were intracardially perfused and the brain tissue was processed for histology. ...
Objective
Sudden unexpected death in epilepsy (SUDEP) is the leading cause of death in patients with refractory epilepsy. Although the mechanisms for SUDEP are incompletely understood, seizure‐induced respiratory arrest (S‐IRA) has been strongly and consistently implicated. A body of evidence indicates that serotonin (5‐HT), a modulator of breathing, plays a critical role in SUDEP. Because the 5‐HT and norepinephrine (NE) systems interact in many biologic processes and NE is known to modulate breathing and seizures, we hypothesized that NE may play a role in S‐IRA and SUDEP.
Methods
We examined the effects of pharmacologic manipulation of 5‐HT and NE on S‐IRA and death following maximal electroshock (MES)–induced seizures in adult wild‐type (WT) mice, genetically 5‐HT neuron–deficient (Lmx1bf/f/p) mice, and chemically NE neuron–deficient mice. Mice were treated with pharmacologic agents targeting the serotonergic and noradrenergic systems and subjected to seizure induction via MES while breathing was measured via whole‐body plethysmography.
Results
S‐IRA and death was reduced in WT mice with NE reuptake inhibitors (NRIs), reboxetine and atomoxetine, selective serotonin reuptake inhibitors (SSRIs), fluoxetine and citalopram, and the dual 5‐HT/NE reuptake inhibitor (SNRI), duloxetine. S‐IRA and death was also reduced in Lmx1bf/f/p mice with reboxetine and fluoxetine. The protective effects of the reuptake inhibitors were prevented by the α1 antagonist, prazosin. Citalopram did not reduce S‐IRA and death in NE neuron–deficient mice.
Significance
These data suggest that 5‐HT and NE critically interact in the modulation of breathing following a seizure and potentially inform preventive strategies for SUDEP.
... Furthermore, there are widespread projections from limbic and non-auditory pathways, such as somatosensory pathways, to the auditory network [17][18][19][20][21][22][23][24][25][26][27]. ...
BACKGROUND
There is increasing evidence from animal and human studies that bimodal neuromodulation combining sound and electrical somatosensory stimulation of the tongue can induce extensive brain changes and treat tinnitus.
OBJECTIVE
The main objectives of the proposed clinical study are to confirm the efficacy, safety, and tolerability of treatment demonstrated in a previous large-scale study of bimodal auditory and trigeminal nerve (tongue) stimulation (Treatment Evaluation of Neuromodulation for Tinnitus - Stage A1); evaluate the therapeutic effects of adjusting stimulation parameters over time; and determine the contribution of different features of bimodal stimulation in improving tinnitus outcomes.
METHODS
This study will be a prospective, randomized, double-blind, parallel-arm, comparative clinical trial of a 12-week treatment for tinnitus using a Conformité Européenne (CE)–marked device with a pre-post and 12-month follow-up design. Four treatment arms will be investigated, in which each arm consists of two different stimulation settings, with the first setting presented during the first 6 weeks and the second setting presented during the next 6 weeks of treatment. The study will enroll 192 participants, split in a ratio of 80:80:16:16 across the four arms. Participants will be randomized to one of four arms and stratified to minimize baseline variability in four categories: two separate strata for sound level tolerance (using loudness discomfort level as indicators for hyperacusis severity), high tinnitus symptom severity based on the Tinnitus Handicap Inventory (THI), and tinnitus laterality. The primary efficacy endpoints are within-arm changes in THI and Tinnitus Functional Index as well as between-arm changes in THI after 6 weeks of treatment for the full cohort and two subgroups of tinnitus participants (ie, one hyperacusis subgroup and a high tinnitus symptom severity subgroup). Additional efficacy endpoints include within-arm or between-arm changes in THI after 6 or 12 weeks of treatment and in different subgroups of tinnitus participants as well as at posttreatment assessments at 6 weeks, 6 months, and 12 months. Treatment safety, attrition rates, and compliance rates will also be assessed and reported.
RESULTS
This study protocol was approved by the Tallaght University Hospital/St. James’s Hospital Joint Research Ethics Committee in Dublin, Ireland. The first participant was enrolled on March 20, 2018. The data collection and database lock are expected to be completed by February 2020, and the data analysis and manuscript submission are expected to be conducted in autumn of 2020.
CONCLUSIONS
The findings of this study will be disseminated to relevant research, clinical, and health services and patient communities through publications in peer-reviewed journals and presentations at scientific and clinical conferences.
CLINICALTRIAL
ClinicalTrials.gov NCT03530306; https://clinicaltrials.gov/ct2/show/NCT03530306
INTERNATIONAL REGISTERED REPORT
DERR1-10.2196/13176
... In addition to the ascending pathway, there are dense descending connections from higher auditory and cognitive centers down to earlier stages of auditory neurons, which provide a way for sound perception to be modified or fine-tuned by attention and learning centers [11][12][13][14][15][16]. Furthermore, there are widespread projections from limbic and nonauditory pathways, such as somatosensory pathways, to the auditory network [17][18][19][20][21][22][23][24][25][26][27]. ...
Background:
There is increasing evidence from animal and human studies that bimodal neuromodulation combining sound and electrical somatosensory stimulation of the tongue can induce extensive brain changes and treat tinnitus.
Objective:
The main objectives of the proposed clinical study are to confirm the efficacy, safety, and tolerability of treatment demonstrated in a previous large-scale study of bimodal auditory and trigeminal nerve (tongue) stimulation (Treatment Evaluation of Neuromodulation for Tinnitus - Stage A1); evaluate the therapeutic effects of adjusting stimulation parameters over time; and determine the contribution of different features of bimodal stimulation in improving tinnitus outcomes.
Methods:
This study will be a prospective, randomized, double-blind, parallel-arm, comparative clinical trial of a 12-week treatment for tinnitus using a Conformité Européenne (CE)-marked device with a pre-post and 12-month follow-up design. Four treatment arms will be investigated, in which each arm consists of two different stimulation settings, with the first setting presented during the first 6 weeks and the second setting presented during the next 6 weeks of treatment. The study will enroll 192 participants, split in a ratio of 80:80:16:16 across the four arms. Participants will be randomized to one of four arms and stratified to minimize baseline variability in four categories: two separate strata for sound level tolerance (using loudness discomfort level as indicators for hyperacusis severity), high tinnitus symptom severity based on the Tinnitus Handicap Inventory (THI), and tinnitus laterality. The primary efficacy endpoints are within-arm changes in THI and Tinnitus Functional Index as well as between-arm changes in THI after 6 weeks of treatment for the full cohort and two subgroups of tinnitus participants (ie, one hyperacusis subgroup and a high tinnitus symptom severity subgroup). Additional efficacy endpoints include within-arm or between-arm changes in THI after 6 or 12 weeks of treatment and in different subgroups of tinnitus participants as well as at posttreatment assessments at 6 weeks, 6 months, and 12 months. Treatment safety, attrition rates, and compliance rates will also be assessed and reported.
Results:
This study protocol was approved by the Tallaght University Hospital/St. James's Hospital Joint Research Ethics Committee in Dublin, Ireland. The first participant was enrolled on March 20, 2018. The data collection and database lock are expected to be completed by February 2020, and the data analysis and manuscript submission are expected to be conducted in autumn of 2020.
Conclusions:
The findings of this study will be disseminated to relevant research, clinical, and health services and patient communities through publications in peer-reviewed journals and presentations at scientific and clinical conferences.
Trial registration:
ClinicalTrials.gov NCT03530306; https://clinicaltrials.gov/ct2/show/NCT03530306.
International registered report identifier (irrid):
DERR1-10.2196/13176.
... After perfusion and serial sectioning, we followed immunohistochemistry (IHC) procedures identical to those used in our previous studies (Hormigo et al. 2012(Hormigo et al. , 2015(Hormigo et al. , 2017. Free-floating sections were incubated with either primary mouse anti-TH (#22941-Diasorin) or rabbit anti-FG (fluorochrome) antibodies for 72 h at 4 °C, and then washed and incubated with their corresponding secondary biotinylated goat anti-mouse or biotinylated anti-rabbit antibodies (#BA-2000-Vector Laboratories) for 2 h at room temperature. ...
... All sections were mounted on slides, and dehydrated and coverslipped with Entellan ® Neu (Merck, Darmstadt, Germany). In addition, several series were counter-stained with either the cresyl violet Nissl staining (Pilati et al. 2008;Hormigo et al. 2012;Zhu et al. 2015) or the NADPH-diaphorase staining (Garcia-Rill 1991;Young et al. 1992) for structural and cytoarchitectonic reference. ...
The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.
... The existing literature on DSP-4 does not offer evidence of non-specific side effects of DSP-4 leading to the development of hyperalgesia. Rather, the literature supports DSP-4 as a disrupter of noradrenergic transmission due to axonal disruption (Ross and Renyl, 1976;Grzanna et al., 1989;Fritschy and Grzanna, 1991;Cheetham et al., 1996;Hormigo et al., 2012) It remains unclear whether increased pain sensitivity in animals treated with DSP-4 is solely due to the loss of descending anti-nociceptive noradrenergic tone and/or a result of chronic injury from to oxidative damage and inflammation in the dorsal horn. Oxidative stress is a widely recognized hallmark of neuropathic pain (Salvemini et al., 2011;Janes et al., 2012;Little et al., 2012a). ...
Central neuropathic pain (CNP) a significant problem for many people, is not well-understood and difficult to manage. Dysfunction of the central noradrenergic system originating in the locus coeruleus (LC) may be a causative factor in the development of CNP. The LC is the major noradrenergic nucleus of the brain and plays a significant role in central modulation of nociceptive neurotransmission. Here, we examined CNS pathophysiological changes induced by intraperotineal administration of the neurotoxin DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride). Administration of DSP-4 decreased levels of norepinephrine in spinal tissue and cerebrospinal fluid (CSF) and led to the development of thermal and mechanical hyperalgesia over 21 days, that was reversible with morphine. Hyperalgesia was accompanied by significant increases in noradrenochrome (oxidized norepinephrine) and expression of 4-hydroxynonenal in CSF and spinal cord tissue respectively at day 21, indicative of oxidative stress. In addition, spinal levels of pro-inflammatory cytokines (interleukins 1β and 6, tumor necrosis factor-α), as well as the the anti-inflammatory cytokine interleukin10 were also significantly elevated at day 21, indicative that an inflammatory response occurred. The inflammatory effect of DSP-4 presented in this study that includes oxidative stress may be particularly useful in elucidating mechanisms of CNP in inflammatory disease states.
... We used 12 animals, 6 males (3 control and 3 DSP-4-injected) and 6 females (3 control and 3 DSP-4-injected), to study the pattern of DBH immunoreactivity in the LC and the cochlear nerve root. After perfusion and serial section protocol, we followed identical immunohistochemistry (IHC) procedures to those used in our previous studies (Hormigo et al. 2012(Hormigo et al. , 2015. The antibodies used and their dilution are shown in Table 1. ...
... One of them is the coerulean projection to the pedunculopontine tegmental nucleus that modulates the primary startle circuit at the level of the pontine reticular nucleus (Jones 1991;Fendt et al. 2001;Alsene and Bakshi 2011). Another one is the coerulean projections to auditory nuclei that are involved in the prepulse inhibition such as the inferior colliculus (Hormigo et al. 2012;Yeomans et al. 2006) and the ventral nucleus of the trapezoid body, which modulates CRN activity (Mulders and Robertson 2001;Gómez-Nieto et al. 2008a, 2014b. In sum, the existence of direct projections from the LC to the CRNs might establish the LC as an essential structure in the neuronal substrates underlying survival and escape behavioral paradigms in response to unexpected loud sounds. ...
The noradrenergic locus coeruleus (LC) plays an important role in the promotion and maintenance of arousal and alertness. Our group recently described coerulean projections to cochlear root neurons (CRNs), the first relay of the primary acoustic startle reflex (ASR) circuit. However, the role of the LC in the ASR and its modulation, prepulse inhibition (PPI), is not clear. In this study, we damaged LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then assessed ASR and PPI in male and female rats. Our results showed that ASR amplitude was higher in males at 14 days after DSP-4 injection when compared to pre-administration values and those in the male control group. Such modifications in ASR amplitude did not occur in DSP-4-injected females, which exhibited ASR amplitude within the range of control values. PPI differences between males and females seen in controls were not observed in DSP-4-injected rats for any interstimulus interval tested. DSP-4 injection did not affect ASR and PPI latencies in either the male or the female groups, showing values that were consistent with the sex-related variability observed in control rats. Furthermore, we studied the noradrenergic receptor system in the cochlear nerve root using gene expression analysis. When compared to controls, DSP-4-injected males showed higher levels of expression in all adrenoceptor subtypes; however, DSP-4-injected females showed varied effects depending on the receptor type, with either up-, downregulations, or maintenance of expression levels. Lastly, we determined noradrenaline levels in CRNs and other LC-targeted areas using HPLC assays, and these results correlated with behavioral and adrenoceptor expression changes post DSP-4 injection. Our study supports the participation of LC in ASR and PPI, and contributes toward a better understanding of sex-related differences observed in somatosensory gating paradigms.
... Once the animals had recovered from the CCI surgery, one group of animals received N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), a noradrenergic neurotoxin (50 mg/kg ip; Sigma, St. Louis, MO, USA) or the vehicle alone Hormigo et al., 2012). In another set of animals, desipramine (DMI, 10 mg/kg/day; Sigma-Aldrich Chemicals, Madrid, Spain) or the vehicle was administered subcutaneously (sc) for two weeks. ...
Previous findings suggest that neuropathic pain induces characteristic changes in the noradrenergic system that may modify the sensorial and affective dimensions of pain. We raise the hypothesis that different drugs that manipulate the noradrenergic system can modify specific domains of pain. In the chronic constriction injury (CCI) model of neuropathic pain, the sensorial (von Frey and acetone tests) and the affective (place escape/avoidance paradigm) domains of pain were evaluated in rats 1 and 2 weeks after administering the noradrenergic neurotoxin [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride] (DSP4, 50 mg/kg). In other animals, we evaluated the effect of enhancing noradrenergic tone in the 2 weeks after injury by administering the antidepressant desipramine (10 mg/kg/day, delivered by osmotic minipumps) during this period, a noradrenaline reuptake inhibitor. Moreover, the phosphorylation of the extracellular signal regulated kinases (p-ERK) in the anterior cingulate cortex (ACC) was also assessed. The ACC receives direct inputs from the main noradrenergic nucleus, the locus coeruleus, and ERK activation has been related with the expression of pain-related negative affect. These studies revealed that DSP4 almost depleted noradrenergic axons in the ACC and halved noradrenergic neurons in the locus coeruleus along with a decrease in the affective dimension and an increased of p-ERK in the ACC. However, it did not modify sensorial pain perception. By contrast, desipramine reduced pain hypersensitivity, while completely impeding the reduction of the affective pain dimension and without modifying the amount of p-ERK. Together results suggest that the noradrenergic system may regulate the sensorial and affective sphere of neuropathic pain independently.
