Reductions in cortical alpha activity, enhancements in neural responses and impaired gap detection caused by sodium salicylate in awake guinea pigs.

Tinnitus chronically affects between 10-15% of the population but, despite its prevalence, the underlying mechanisms are still not properly understood. One experimental model involves administration of high doses of sodium salicylate, as this is known to reliably induce tinnitus in both humans and animals. Guinea pigs were implanted with chronic electrocorticography (ECoG) electrode arrays, with silver-ball electrodes placed on the dura over left and right auditory cortex. Two more electrodes were positioned over the cerebellum to monitor auditory brainstem responses (ABRs). We recorded resting-state and auditory evoked neural activity from awake animals before and 2 h following salicylate administration (350 mg/kg; i.p.). Large increases in click-evoked responses (> 100%) were evident across the whole auditory cortex, despite significant reductions in wave I ABR amplitudes (in response to 20 kHz tones), which are indicative of auditory nerve activity. In the same animals, significant decreases in 6-10 Hz spontaneous oscillations (alpha waves) were evident over dorsocaudal auditory cortex. We were also able to demonstrate for the first time that cortical evoked potentials can be inhibited by a preceding gap in background noise [gap-induced pre-pulse inhibition (PPI)], in a similar fashion to the gap-induced inhibition of the acoustic startle reflex that is used as a behavioural test for tinnitus. Furthermore, 2 h following salicylate administration, we observed significant deficits in PPI of cortical responses that were closely aligned with significant deficits in behavioural responses to the same stimuli. Together, these data are suggestive of neural correlates of tinnitus and oversensitivity to sound (hyperacusis).

Neural changes accompanying tinnitus following unilateral acoustic trauma in the guinea pig.

Animal models of tinnitus allow us to study the relationship between changes in neural activity and the tinnitus percept. Here, guinea pigs were subjected to unilateral noise trauma and tested behaviourally for tinnitus 8 weeks later. By comparing animals with tinnitus with those without, all of which were noise-exposed, we were able to identify changes unique to the tinnitus group. Three physiological markers known to change following noise exposure were examined: spontaneous firing rates (SFRs) and burst firing in the inferior colliculus (IC), evoked auditory brainstem responses (ABRs), and the number of neurons in the cochlear nucleus containing nitric oxide synthase (NOS). We obtained behavioural evidence of tinnitus in 12 of 16 (75%) animals. Both SFRs and incidences of burst firing were elevated in the IC of all noise-exposed animals, but there were no differences between tinnitus and no-tinnitus animals. There were significant decreases in ipsilateral ABR latencies in tinnitus animals, contrary to what might be expected with a small hearing loss. Furthermore, there was an ipsilateral-contralateral asymmetry in NOS staining in the ventral cochlear nucleus (VCN) that was only apparent in tinnitus animals. Tinnitus animals had a significantly greater number of NOS-containing neurons on the noise-exposed side, whereas no-tinnitus animals did not. These data suggest that measuring NOS in the VCN and recording ABRs supplement behavioural methods for confirming tinnitus in animals, and that nitric oxide is involved in plastic neural changes associated with tinnitus.

Progesterone is neuroprotective following cerebral ischaemia in reproductively ageing female mice.

Gender differences in both vulnerability to stroke and outcome following cerebral ischaemia have frequently been observed and attributed to the action of steroid hormones. Progesterone is a candidate neuroprotective factor for stroke; however, studies are lacking which: (i) study those groups representing high risk i.e. postmenopausal females; (ii) administer progesterone solely post-ischaemia; and (iii) combine histopathological and functional assessments. Postmenopausal females, along with males, represent the group at highest risk of cerebral stroke and can be modelled using aged or ovariectomized animals. In the current study, we aimed to determine the neuroprotective effects of progesterone administration following cerebral ischaemia in aged and ovariectomized mice. Following transient middle cerebral artery occlusion, progesterone was administered at 1, 6 and 24 h post-ischaemia to aged and ovariectomized female mice. At 48 h post-ischaemia, progesterone significantly reduced the lesion volume (P < 0.05) but had no effect on neurological outcome in aged female mice. Whereas in ovariectomized mice, at 48 h post-ischaemia, progesterone treatment had no effect on the amount of lesion volume present but did significantly improve neurological outcome. In a further study of ovariectomized mice, allowed to survive for 7 days post-ischaemia, progesterone treatment significantly improved motor outcome as assessed using both the rotarod and grid test. In fact, by 7 days post-ischaemia, progesterone-treated ovariectomized mice did not differ significantly in performance compared with shams, whereas vehicle-treated ovariectomized mice displayed a significant functional impairment following ischaemia. The current study has demonstrated that progesterone has different neuroprotective effects whether it is administered to aged or ovariectomized female mice and emphasizes the need to combine histopathological and functional outcomes within the same study. In addition, as progesterone-only treatment may not improve all outcomes in all groups, therapies that combine progesterone with other neuroprotective candidates should be investigated to maximize benefit following stroke.

Assessing cortico-hippocampal functional connectivity under anesthesia and kainic acid using generalized partial directed coherence.

A significant challenge in modern neuroscience lies in determining the functional connectivity between discrete populations of neurones and brain regions. In this study, a variation of partial directed coherence, the generalized partial directed coherence (gPDC), along with a newly proposed critical value for gPDC, were applied on recorded local field potentials (LFPs) and single-unit activity, in order to assess information flow between medial prefrontal cortex (mPFC) and hippocampus and within the hippocampus of the rat brain, under isoflurane anesthesia and kainic acid-induced enhanced neuronal activity. Our findings suggest that, under anesthesia, there exists a continuous information flow from hippocampus towards mPFC, reversed mostly during activity bursts occurring in the mPFC. Moreover, there was a clear directional connection from the lateral towards medial dorsal hippocampus, most prominent in the beta frequency band (10-30 Hz). Kainic acid resulted in partially disrupting the reciprocal cortico-hippocampal connectivity and reversing the intra-hippocampal one. The biological implications of these findings on the effects of anesthesia and kainic acid in brain connectivity, along with implementation issues of gPDC analysis on field potentials and spike trains, are extensively discussed.

Is progesterone a candidate neuroprotective factor for treatment following ischemic stroke?

Gender differences in stroke outcome have implicated steroid hormones as potential neuroprotective candidates. However, no clinical trials examining hormone replacement therapy on outcome following ischemic stroke have investigated the effect of progesterone-only treatment. In this review the authors examine the experimental evidence for the neuroprotective potential of progesterone and give an insight into potential mechanisms of action following ischemic stroke. To date, 17 experimental studies have investigated the neuroprotective potential of progesterone for ischemic stroke in terms of ability to both reduce cell loss and increase functional outcome. Of these 17 published studies the majority reported a beneficial effect with three studies reporting a nil effect and only one study reporting a negative effect. However, there are important issues that the authors address in this review in terms of the methodological quality of studies in relation to the STAIR recommendations. In terms of the proposed mechanisms of progesterone neuroprotection we show that progesterone is versatile and acts at multiple targets to facilitate neuronal survival and minimize cell damage and loss. A large amount of experimental evidence indicates that progesterone is a neuroprotective candidate for ischemic stroke; however, to progress to clinical trial a number of key experimental studies remain outstanding.

Inhibition of endocannabinoid metabolism attenuates enhanced hippocampal neuronal activity induced by kainic acid.

The endogenous cannabinoid system regulates neuronal excitability. The effects of inhibiting fatty acid amide hydrolase (FAAH), the enzyme responsible for metabolism of the endocannabinoid anandamide, on kainic acid (KA)-induced neuronal activity were investigated in the rat in vivo, using the selective FAAH inhibitor URB597. Hippocampal neuronal ensemble unit activity was recorded in isoflurane-anesthetized rats using 16-wire microelectrode arrays. Separate groups of rats were administered with single doses of KA alone, KA and URB597 (0.3 or 1 mg kg(-1), i.p.), or URB597 (1 mg kg(-1)) alone. The role of the cannabinoid CB1 receptor in mediating the effects of URB597 was explored using the CB1 selective antagonists AM251, either alone or prior to KA and URB597 (1 mg kg(-1)) administration, and SR141716A, administered prior to KA and URB597 (1 mg kg(-1)). Neuronal firing and burst firing rates were examined in animals with confirmed dorsal hippocampal placements. KA induced an increase in both firing and burst firing rates, effects which were attenuated by URB597 in a dose-related manner. Pretreatment with AM251 or SR141716A partly attenuated the URB597-mediated effects on firing and burst firing rate. Rats treated with AM251 or URB597 alone did not exhibit any significant change in either firing or burst firing rates compared with basal activity. These results suggest that the inhibition of endocannabinoid metabolism can suppress hyperexcitability in the rat hippocampus, partly via a CB1 receptor-mediated mechanism.

Gap-induced reductions of evoked potentials in the auditory cortex: A possible objective marker for the presence of tinnitus in animals.

Animal models of tinnitus are essential for determining the underlying mechanisms and testing pharmacotherapies. However, there is doubt over the validity of current behavioural methods for detecting tinnitus. Here, we applied a stimulus paradigm widely used in a behavioural test (gap-induced inhibition of the acoustic startle reflex GPIAS) whilst recording from the auditory cortex, and showed neural response changes that mirror those found in the behavioural tests. We implanted guinea pigs (GPs) with electrocorticographic (ECoG) arrays and recorded baseline auditory cortical responses to a startling stimulus. When a gap was inserted in otherwise continuous background noise prior to the startling stimulus, there was a clear reduction in the subsequent evoked response (termed gap-induced reductions in evoked potentials; GIREP), suggestive of a neural analogue of the GPIAS test. We then unilaterally exposed guinea pigs to narrowband noise (left ear; 8-10 kHz; 1 h) at one of two different sound levels - either 105 dB SPL or 120 dB SPL - and recorded the same responses seven-to-ten weeks following the noise exposure. Significant deficits in GIREP were observed for all areas of the auditory cortex (AC) in the 120 dB-exposed GPs, but not in the 105 dB-exposed GPs. These deficits could not simply be accounted for by changes in response amplitudes. Furthermore, in the contralateral (right) caudal AC we observed a significant increase in evoked potential amplitudes across narrowband background frequencies in both 105 dB and 120 dB-exposed GPs. Taken in the context of the large body of literature that has used the behavioural test as a demonstration of the presence of tinnitus, these results are suggestive of objective neural correlates of the presence of noise-induced tinnitus and hyperacusis.

Effects of the cannabinoid CB1 agonist ACEA on salicylate ototoxicity, hyperacusis and tinnitus in guinea pigs.

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2'-chloroethylamide (ACEA), a highly-selective CB1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6-10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis.

Tinnitus-related changes in the inferior colliculus.

Tinnitus is highly complex, diverse, and difficult to treat, in part due to the fact that the underlying causes and mechanisms remain elusive. Tinnitus is generated within the auditory brain; however, consolidating our understanding of tinnitus pathophysiology is difficult due to the diversity of reported effects and the variety of implicated brain nuclei. Here, we focus on the inferior colliculus (IC), a midbrain structure that integrates the vast majority of ascending auditory information and projects via the thalamus to the auditory cortex. The IC is also a point of convergence for corticofugal input and input originating outside the auditory pathway. We review the evidence, from both studies with human subjects and from animal models, for the contribution the IC makes to tinnitus. Changes in the IC, caused by either noise exposure or drug administration, involve fundamental, heterogeneous alterations in the balance of excitation and inhibition. However, differences between hearing loss-induced pathology and tinnitus-related pathology are not well understood. Moreover, variability in tinnitus induction methodology has a significant impact on subsequent neural and behavioral changes, which could explain some of the seemingly contradictory data. Nonetheless, the IC is likely involved in the generation and persistence of tinnitus perception.

Modulating central gain in tinnitus: changes in nitric oxide synthase in the ventral cochlear nucleus.

A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators - components of neural signaling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO) - a free radical and potent neuromodulator in the mammalian brain - that regulates plasticity via both pre-synaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN). Here, we examined nitric oxide synthase (NOS) - the enzyme responsible for NO production - in the guinea pig VCN following acoustic trauma. NOS was present in most cell types - including spherical and globular bushy cells, small, medium, and large multipolar cells, and octopus cells - spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE) resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioral evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as 1 day after AOE and was also present in some animals at 3, 7, and 21 days after AOE. However, it was not until 8 weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise exposure.

Changes in the response properties of inferior colliculus neurons relating to tinnitus.

Tinnitus is often identified in animal models by using the gap prepulse inhibition of acoustic startle. Impaired gap detection following acoustic over-exposure (AOE) is thought to be caused by tinnitus "filling in" the gap, thus, reducing its salience. This presumably involves altered perception, and could conceivably be caused by changes at the level of the neocortex, i.e., cortical reorganization. Alternatively, reduced gap detection ability might reflect poorer temporal processing in the brainstem, caused by AOE; in which case, impaired gap detection would not be a reliable indicator of tinnitus. We tested the latter hypothesis by examining gap detection in inferior colliculus (IC) neurons following AOE. Seven of nine unilaterally noise-exposed guinea pigs exhibited behavioral evidence of tinnitus. In these tinnitus animals, neural gap detection thresholds (GDTs) in the IC significantly increased in response to broadband noise stimuli, but not to pure tones or narrow-band noise. In addition, when IC neurons were sub-divided according to temporal response profile (onset vs. sustained firing patterns), we found a significant increase in the proportion of onset-type responses after AOE. Importantly, however, GDTs were still considerably shorter than gap durations commonly used in objective behavioral tests for tinnitus. These data indicate that the neural changes observed in the IC are insufficient to explain deficits in behavioral gap detection that are commonly attributed to tinnitus. The subtle changes in IC neuron response profiles following AOE warrant further investigation.

A novel behavioural approach to detecting tinnitus in the guinea pig.

Tinnitus, the perception of sound in the absence of an external stimulus, is a particularly challenging condition to demonstrate in animals. In any animal model, objective confirmation of tinnitus is essential before we can study the neural changes that produce it. A gap detection method, based on prepulse inhibition of the whole-body startle reflex, is often used as a behavioural test for tinnitus in rodents. However, in the guinea pig the whole-body startle reflex is subject to rapid habituation and hence is not an ideal behavioural measure. By contrast, in this species the Preyer or pinna reflex is a very reliable indicator of the startle response and is much less subject to habituation. We have developed a novel adaptation of the gap detection paradigm, which uses the Preyer reflex to measure the startle response, rather than whole-body movement. Using this method, we have demonstrated changes in gap detection, in guinea pigs where tinnitus had been induced by the administration of a high dose of salicylate. Our data indicate that the Preyer reflex gap detection method is a reliable test for tinnitus in guinea pigs.

Effect of pre-ischaemic conditioning on hypoxic depolarization of dopamine efflux in the rat caudate brain slice measured in real-time with fast cyclic voltammetry.

Fast cyclic voltammetry can be used to measure dopamine release after oxygen and glucose deprivation (OGD) induced anoxic depolarization in vitro. Here we measure dopamine efflux with 1s time resolution, which is appropriate to measure OGD-evoked dopamine efflux accurately. In the present study, we examined whether OGD-evoked dopamine efflux could be used to show pre-ischaemic conditioning in the rat caudate brain slice. Caudate slices were exposed to 0, 2, or 10 min OGD pre-ischaemic conditioning, then 60 min later exposed to a second OGD event of 15 min duration. We measured the OGD-evoked dopamine efflux using fast cyclic voltammetry and in some experiments caudate dopamine and DOPAC tissue levels were measured using HPLC and 20 µm cryostat sections were Nissl stained to indicate neuronal loss. We found that 10 but not 2 min OGD pre-ischaemic conditioning resulted in a longer time to onset of OGD-evoked dopamine efflux on the main OGD event (475 ± 31 and 287 ± 30 s for 10 Vs 0 min pre-ischaemic conditioning respectively). Further, 10 min OGD pre-ischaemic conditioning resulted in less dopamine efflux on the second OGD event (4.23 ± 1.12 and 8.14 ± 0.82 µM for 10 Vs 0 min pre-ischaemic conditioning respectively), despite these slices having similar tissue dopamine content and DOPAC/DA ratio, and the rate of dopamine release was slower in the main OGD event (21 ± 5 and 74 ± 8 nM/s for 10 Vs 0 min pre-ischaemic conditioning respectively). These data suggest that 10 min OGD pre-ischaemic conditioning can evoke tolerance to a second OGD event and that voltammetric recording of OGD-evoked dopamine efflux is a useful model of pre-ischaemic conditioning in neuronal tissue.

Cortical inactivation by cooling in small animals.

Reversible inactivation of the cortex by surface cooling is a powerful method for studying the function of a particular area. Implanted cooling cryoloops have been used to study the role of individual cortical areas in auditory processing of awake-behaving cats. Cryoloops have also been used in rodents for reversible inactivation of the cortex, but recently there has been a concern that the cryoloop may also cool non-cortical structures either directly or via the perfusion of blood, cooled as it passed close to the cooling loop. In this study we have confirmed that the loop can inactivate most of the auditory cortex without causing a significant reduction in temperature of the auditory thalamus or other subcortical structures. We placed a cryoloop on the surface of the guinea pig cortex, cooled it to 2°C and measured thermal gradients across the neocortical surface. We found that the temperature dropped to 20-24°C among cells within a radius of about 2.5 mm away from the loop. This temperature drop was sufficient to reduce activity of most cortical cells and led to the inactivation of almost the entire auditory region. When the temperature of thalamus, midbrain, and middle ear were measured directly during cortical cooling, there was a small drop in temperature (about 4°C) but this was not sufficient to directly reduce neural activity. In an effort to visualize the extent of neural inactivation we measured the uptake of thallium ions following an intravenous injection. This confirmed that there was a large reduction of activity across much of the ipsilateral cortex and only a small reduction in subcortical structures.

Sustained levels of progesterone prior to the onset of cerebral ischemia are not beneficial to female mice.

Female gender, which is abolished following ovariectomy and reproductive senescence, is associated with improved outcome following cerebral stroke. Estrogen replacement partially restores this benefit of the female gender but the effect of progesterone in hormone-deficient animals is currently unknown. We evaluated various outcomes following middle cerebral artery occlusion (MCAO) in ovariectomised female mice, with a physiologically relevant restoration of progesterone levels. Ovariectomised female mice had significantly elevated plasma (P=<0.05) and brain progesterone levels (P=<0.01) following implantation of a 21-day release pellet (50mg) compared with mice that received placebo implants 7 days prior to undergoing 60 min MCAO. Assessment of well-being (body weight recovery) and neurological score at 24h and 48h post-MCAO indicated that MCAO significantly worsened outcome compared with sham-operated mice but progesterone had no effect. MCAO resulted in a substantial lesion formation and a significant increase (P<0.05) in ipsilateral brain water content, both of which were not affected by progesterone treatment. Furthermore, there was no significant alteration in ipsilateral Aquaporin-4 (AQP4) expression following MCAO or progesterone treatment. The present study indicates that sustained physiologically relevant levels of progesterone prior to cerebral ischemia neither benefited nor worsened outcomes in previously ovariectomised female mice.