Suppression of putative tinnitus-related activity by extra-cochlear electrical stimulation.

Studies on animals have shown that noise-induced hearing loss is followed by an increase of spontaneous firing at several stages of the central auditory system. This central hyperactivity has been suggested to underpin the perception of tinnitus. It was shown that decreasing cochlear activity can abolish the noise-induced central hyperactivity. This latter result further suggests that an approach consisting of reducing cochlear activity may provide a therapeutic avenue for tinnitus. In this context, extra-cochlear electric stimulation (ECES) may be a good candidate to modulate cochlear activity and suppress tinnitus. Indeed, it has been shown that a positive current applied at the round window reduces cochlear nerve activity and can suppress tinnitus reliably in tinnitus subjects. The present study investigates whether ECES with a positive current can abolish the noise-induced central hyperactivity, i.e., the putative tinnitus-related activity. Spontaneous and stimulus-evoked neural activity before, during and after ECES was assessed from single-unit recordings in the inferior colliculus of anesthetized guinea pigs. We found that ECES with positive current significantly decreases the spontaneous firing rate of neurons with high characteristic frequencies, whereas negative current produces the opposite effect. The effects of the ECES are absent or even reversed for neurons with low characteristic frequencies. Importantly, ECES with positive current had only a marginal effect on thresholds and tone-induced activity of collicular neurons, suggesting that the main action of positive current is to modulate the spontaneous firing. Overall, cochlear electrical stimulation may be a viable approach for suppressing some forms of (peripheral-dependent) tinnitus.

Bedaquiline- and clofazimine- selected Mycobacterium tuberculosis mutants: further insights on resistance driven largely by Rv0678.

Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain in Mycobacterium tuberculosis (Mtb). While mutations in the BDQ target gene, atpE, are rare in clinical isolates, mutations in the Rv0678 gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5, are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations in Rv0678, atpE and pepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations in Rv0678, atpE or pepQ, while CFZ-exposed isolates presented mutations in Rv0678 and pepQ, but none in atpE. Twenty-eight Rv0678 mutations had previously been described among in vitro selected mutants or in patients' isolates, while 85 were new. Mutations were scattered across the Rv0678 gene without apparent hotspot. While most Rv0678 mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in the Rv0678 start codon. Finally, in silico characterization of Rv0678 mutations suggests that especially the C46R mutant may render Rv0678 less stable.

Spontaneous firing patterns in the medial geniculate nucleus in a guinea pig model of tinnitus.

The mechanism of tinnitus, the perception of sound in the absence of acoustic stimulation, remains as yet unknown. It has been proposed that tinnitus is caused by altered spontaneous activity in the auditory pathway following cochlear damage in combination with inadequate gating at the level of the auditory thalamus, the medial geniculate nucleus (MGN). To investigate this further we made electrophysiological recordings in MGN of guinea pigs (n = 9) with and without tinnitus after acoustic trauma (continuous loud tone at 10 kHz, 124 dB SPL for 2 h). Parameters of interest were spontaneous tonic and burst firing. After acoustic trauma, 5 out of 9 guinea pigs developed signs of tinnitus as determined by the gap prepulse inhibition of acoustic startle. Spontaneous firing rates were significantly increased in the tinnitus animals as compared to the non-tinnitus animals and this change was specific to pure-tone responsive MGN neurons. However, burst firing parameters, including number of bursts per minute, burst duration, number of spikes in each burst, and percentage of spikes occurring in a burst, were not different between tinnitus and non-tinnitus animals. In addition, our data showed a strong dependence of spontaneous firing rates with heart rate, which implies that monitoring physiological status in animals is pertinent to obtaining reliable data when recording at higher levels of the auditory pathway. Our results suggest that increases in the tonic spontaneous fining rate of pure-tone responsive MGN neurons but not changes in burst firing parameters, are a robust neural signature of tinnitus in anaesthetised animals.

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial.

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

First-line tuberculosis treatment with double-dose rifampicin is well tolerated.

OBJECTIVE: To compare the occurrence of unfavourable treatment and safety outcomes of double-dose rifampicin (RMP; 20 mg/kg/d, intervention) with standard dose (10 mg/kg/d, control) in a first-line tuberculosis (TB) treatment regimen for smear-positive TB patients in Bangladesh.DESIGN: This was a randomised clinical trial. The primary efficacy and safety endpoints were the occurrence of an unfavourable treatment outcome (death, failure, relapse or loss to follow-up) and the occurrence of any serious drug-related adverse event (SAE).RESULTS: In primary efficacy analysis, among 343 control and 347 intervention patients, respectively 15.5% and 11.8% had an unfavourable outcome. In safety analysis, among 349 intervention and 352 control patients, respectively 4.3% and 2.6% experienced an SAE. These differences were not significant. There was a significantly lower occurrence of SAEs, explained by a lower occurrence of hepatic toxicity, in a RMP double-dosed but erroneously HZE (isoniazid+pyrazinamide+ethambutol) under-dosed subgroup.CONCLUSIONS: Our findings show that there is no statistically significant difference in terms of efficacy and safety between standard and double-dose RMP. An accidental finding (related to dosage levels of the standard regimen) suggests that high-dose RMP is potentially a lesser cause of hepatotoxicity. Larger trials with more power, or trials with at least a triple-dose might be needed to clearly see the effect of high-dose RMP on unfavourable outcomes.

Changes in auditory thalamus neural firing patterns after acoustic trauma in rats.

Tinnitus is an abnormal phantom perception associated with cochlear trauma, and is thought to cause changes in the rates and patterns of firing neurons in the central auditory pathway. Recent studies have suggested a key role for the auditory thalamus, the medial geniculate nucleus (MGN), in the generation of tinnitus as it may serve a gating function for information en route to cortex. Dysfunctional gating would lead to abnormal activity reaching cortex and hence inappropriate perception, tinnitus, would occur. In this study we compared spontaneous MGN firing rates and burst firing parameters in Wistar rats with and without behavioural evidence of tinnitus following an acoustic trauma. Data were also compared with animals subjected to sham surgery and at an early time-point (2 weeks) after acoustic trauma. Acoustic trauma resulted in a temporary but not a permanent threshold loss and no differences were found in spontaneous firing rate between any of the groups. However, acoustic trauma, whether resulting in tinnitus or not, was accompanied by a significant decrease in the percentage of neurons showing burst firing. In bursting neurons, the number of spikes occurring in a burst and the number of burst per minutes was also significantly reduced compared to the sham group. Our results show that in our rat model without permanent threshold loss, elevated spontaneous firing rates are not associated with acoustic trauma and/or tinnitus and that burst firing parameters are associated with acoustic trauma but are not a neural signature for tinnitus.

Effects of centrifugal pathways on responses of cochlear nucleus neurons to signals in noise.

The medial olivocochlear (MOC) system, which originates in the brainstem and projects to the outer hair cells in the cochlea, is thought to be involved in improving signal detection in noisy backgrounds. This proposition arises from the observation that the input-output functions of auditory primary afferent fibres to pure tones recorded in a continuous background noise are unmasked by MOC activation, improving the dynamic range, and is supported by both animal and human behavioural experiments. However, it is not known how the unmasking effects observed in the cochlea are translated into higher auditory brain centres, such as the cochlear nucleus, where intrinsic circuitry can potentially modulate any effect. In this study we have investigated the effects of continuous background noise without and with MOC system activation, on responses of different neuron types in the ventral cochlear nucleus of the guinea pig. Results show that the unmasking effects of MOC system activation on tone responses in continuous background noise are present in the cochlear nucleus. These unmasking effects manifest themselves as decompression of input-output functions as well as an improved slope, which results in an improvement in intensity discrimination of the tones. The data show, however, that the strength of the unmasking effects of MOC system activation varies between the different neuronal types. Unmasking was not detected in onset chopper neurons despite its demonstrable presence in other neuronal types in the same animals. These observations may reflect the level of involvement of different neuronal types in intensity discrimination.

Medial geniculate neurons show diverse effects in response to electrical stimulation of prefrontal cortex.

Phantom perceptions have been proposed to arise due to dysfunctional sensory gating at the level of the thalamus. Recently, it has been suggested that tinnitus, a phantom perception of sound, may arise from altered cortico-limbic circuitry and its connection with the auditory thalamus, the medial geniculate nucleus (MGN). Indeed, some elements of this cortico-limbic circuitry, such as the prefrontal cortex (PFC), as well as elements of the auditory pathway, have been shown to be altered in humans with tinnitus. However, the functional connectivity between PFC and MGN has not yet been explored. We therefore investigated the effects of activation of the PFC on neuronal activity in MGN in normal anaesthetized Wistar rats. Bipolar electrical stimulation was delivered to the PFC while recording single neuron activity in the MGN. The majority (81%) of MGN neurons sampled showed a change in their spontaneous firing rate in response to electrical stimulation of the PFC. The effects observed varied greatly between neurons and included combinations of inhibitory and excitatory effects with a wide range of latencies. The effects were not dependent on acoustic response type or MGN subdivision. These data demonstrate that PFC activation can modulate MGN neuronal activity and this connection could potentially play a role in sensory gating of auditory signals.

Effects of pulsatile electrical stimulation of the round window on central hyperactivity after cochlear trauma in guinea pig.

Partial hearing loss induced by acoustic trauma has been shown in animal models to result in an increased spontaneous firing rate in central auditory structures. This so-called hyperactivity has been suggested to be involved in the generation of tinnitus, a phantom auditory sensation. Although there is no universal cure for tinnitus, electrical stimulation of the cochlea, as achieved by a cochlear implant, can result in significant reduction of the tinnitus percept. However, the mechanism by which this tinnitus suppression occurs is as yet unknown and furthermore cochlear implantation may not be an optimal treatment option for tinnitus sufferers who are not profoundly deaf. A better understanding of the mechanism of tinnitus suppression by electrical stimulation of the cochlea, may lead to the development of more specialised devices for those for whom a cochlear implant is not appropriate. This study aimed to investigate the effects of electrical stimulation in the form of brief biphasic shocks delivered to the round window of the cochlea on the spontaneous firing rates of hyperactive inferior colliculus neurons following acoustic trauma in guinea pigs. Effects during the stimulation itself included both inhibition and excitation but spontaneous firing was suppressed for up to hundreds of ms after the cessation of the shock train in all sampled hyperactive neurons. Pharmacological block of olivocochlear efferent action on outer hair cells did not eliminate the prolonged suppression observed in inferior colliculus neurons, and it is therefore likely that activation of the afferent pathways is responsible for the central effects observed.

Catecholaminergic innervation of guinea pig superior olivary complex.

In mammals, olivocochlear neurons in the superior olivary complex project to the cochlea, providing input to outer hair cells and auditory afferents contacting inner hair cells. In the rat it has been demonstrated that olivocochlear neurons receive noradrenergic input, arising from the locus coeruleus and it has been demonstrated in this species using in vitro brain slices that noradrenaline exerts a direct, mostly excitatory effect on an olivocochlear subpopulation. The guinea pig is a more commonly used animal in auditory physiology than the rat and anatomical data on noradrenaline in the auditory brainstem in this species are lacking. Because it has been shown that a compact locus coeruleus is not present in the guinea pig, subtle species differences might be expected. Therefore, using immunohistochemical and tracing techniques we have investigated in the guinea pig (1) the noradrenergic and dopaminergic innervation of the superior olivary complex, (2) the anatomical relationship between noradrenergic fibres and olivocochlear neurons and (3) the origin of the noradrenergic input to this brainstem region. The results show that the guinea pig superior olivary complex receives moderately dense noradrenergic innervation and no dopaminergic innervation. In addition, noradrenergic fibres and varicosities were observed in close contact with both somata and dendrites of olivocochlear neurons, strongly suggestive of synaptic contacts. Finally the results show that a significant component of the noradrenergic innervation of the guinea pig superior olivary complex arises in the locus subcoeruleus, which is a structure likely to be the homologue of the locus coeruleus in rats and other species.

Noradrenergic modulation of brainstem nuclei alters cochlear neural output.

The peripheral auditory sense organ, the cochlea, receives innervation from lateral and medial olivocochlear neurons in the brainstem. These neurons are able to modulate cochlear neural output. Anatomical studies have shown that one of the neurotransmitters which is present in varicosities surrounding the olivocochlear neurons in the brainstem is noradrenaline and previous work on brainstem slices has demonstrated a generally excitatory effect of noradrenaline on medial olivocochlear neurons. In order to assess in vivo the function of the noradrenergic inputs to olivocochlear neurons, we injected noradrenaline in the brainstem of anaesthetised guinea pigs and recorded ipsilateral cochlear electrical activity. Injections of noradrenaline close to the lateral olivocochlear neurons evoked increases in the sound-driven neural activity from the cochlea, measured as compound action potential (CAP) amplitude, as well as in the spontaneous activity, measured as amplitude of the 900 Hz peak of the spectrum of the neural noise in the cochlear fluids. In contrast, noradrenaline in the vicinity of the medial olivocochlear neurons evoked inhibitory effects on both the CAP amplitude and 900 Hz peak. These results indicate most likely an excitatory action of noradrenaline on both the lateral and medial olivocochlear neurons in the brainstem, and show that such noradrenergic inputs can modulate cochlear function.

Modulation of medial geniculate nucleus neuronal activity by electrical stimulation of the nucleus accumbens.

Dysfunctional sensory gating has been proposed to result in the generation of phantom perceptions. In agreement, it has been recently suggested that tinnitus, a phantom perception of sound commonly associated with hearing loss, is the result of a breakdown of circuitry involving the limbic system and the medial geniculate nucleus (MGN) of the thalamus. In humans with tinnitus, structural changes and abnormal activity have been found to occur in the auditory pathway as well as parts of the limbic system such as the nucleus accumbens (NAc). However, at present, no studies have been conducted on the influence of the NAc on the MGN. We investigated the functional connectivity between the NAc and MGN single neurons. Bipolar electrical stimulation was delivered to the NAc while recording single neuron activity in MGN in anesthetized Wistar rats. Histological analysis was used to confirm placement of electrodes. NAc electrical stimulation generally decreased spontaneous firing rates in MGN neurons and, in a limited number of neurons, caused an increase in firing rate. This suggests that NAc can modulate the activity of auditory neurons in the MGN and may play a role in the development of tinnitus.

The thin-layer agar method for direct phenotypic detection of multi- and extensively drug-resistant tuberculosis.

BACKGROUND: Molecular techniques rapidly detect resistance to rifampicin (RMP) and isoniazid (INH), but do not eliminate the need for culture-based drug susceptibility testing (DST) against other drugs. The thin-layer agar (TLA) test, a non-commercial direct DST method, has demonstrated good performance for INH and RMP; however, evidence is still limited, and its applicability for DST of ofloxacin (OFX) and kanamycin (KM) is unknown. DESIGN: We compared 279 TLA DST results with those of MGIT for INH and RMP, and 280 results for OFX and KM with those of the 7H11 agar proportion method, obtained from 320 smear-positive samples from 165 Georgian TB patients. Discrepancies were solved by comparison with a composite reference standard. The prevalence of multidrug-resistant tuberculosis (TB) was 30 of 164 patients (18.3%), 2 (6.7%) of whom had extensively drug-resistant TB. RESULTS: TLA showed 94.7%, 98.2%, 100% and 78.9% sensitivity, respectively, for INH, RMP, OFX and KM, with 100% specificity. Average time to results was 7 days in TLA, 23 in MGIT and 49 for 7H11 agar. CONCLUSIONS: In low-resource settings, TLA can be applied for the rapid detection of resistance to INH, RMP and fluoroquinolones. Further studies are necessary to improve sensitivity to KM and further assess its performance for OFX and other drugs and its applicability in field conditions.

Neuron numbers in the presubiculum, parasubiculum, and entorhinal area of the rat.

Estimates of neuron numbers have been useful in studies of neurodegenerative disorders, and in their animal models, and in the computational modeling of hippocampal function. Although the retrohippocampal region (presubiculum, parasubiculum, and entorhinal area) is an integral part of the hippocampal circuitry and is affected selectively in a number of disorders, estimates of neuron numbers in the rat retrohippocampal region have yet to be published. Such data are necessary ingredients for computational models of the function of this region and will also facilitate a comparison of this region in rats and primates, which will help to determine how well we may expect rat models to predict function and dysfunction in primate brains. In the present study, we used the optical fractionator to estimate the number of neurons in the rat retrohippocampal region. The following estimates were obtained: 3.3 x 10(5) in presubicular layers II and III, 1.5 x 10(5) in parasubicular layers II and III, 2.2 x 10(5) in the combined pre- and parasubicular layers V and VI, 6.6 x 10(4) in medial entorhinal area (MEA) layer II, 1.3 x 10(5) in MEA layer III, 1.9 x 10(5) in MEA layers V and VI, 4.6 x 10(4) in lateral entorhinal area (LEA) layer II, 1.2 x 10(5) in LEA layer III, and 1.4 x 10(5) in LEA layers V and VI. A surprising finding was the large numbers of neurons in the pre- and parasubiculum, which indicate an important role of these areas in the control of the entorhino-hippocampal projection. A comparison of the numbers of neurons in the hippocampus and entorhinal areas in rats with similar estimates in humans revealed that gross input-output relations are largely conserved. Differences between rats and humans may be accounted for by more prominent entorhino-neocortical projections in primates and consequent increases in the number of neurons in the hippocampus and retrohippocampal region, which are dedicated to these projections.

The substantia innominata complex and the peripeduncular nucleus in orofacial dyskinesia: a pharmacological and anatomical study in cats.

It has been shown that orofacial dyskinesia, i.e. a syndrome of abnormal involuntary movements of the facial muscles, can be elicited from the sub-commissural part of the globus pallidus and the adjoining dorsal parts of the extended amygdala in cats. Until now it is unknown whether the peripeduncular nucleus, which receives input from these structures according to anterograde tracing studies, plays a role in the funneling of orofacial dyskinesia to lower output stations. In the present study the connection of the subcommissural part of the globus pallidus and dorsal parts of the extended amygdala with the peripeduncular nucleus was investigated anatomically, using cholera toxin subunit B as a retrograde tracer, and functionally, using intracerebral injections of GABAergic compounds. The anatomical data show that the sub-commissural part of the globus pallidus and dorsal parts of the extended amygdala were marked by cholera toxin sub-unit B-immunoreactive cells following injections of this retrograde tracer into the peripeduncular nucleus. Thus, it could be confirmed that the peripeduncular nucleus receives input from the sub-commissural part of the globus pallidus and dorsal parts of the extended amygdala. Still, the orofacial dyskinesia elicited by local injections of the GABA antagonist picrotoxin (500 ng/0.5 microliters) into the sub-commissural part of the globus pallidus and dorsal extended amygdala was only in part attenuated by local injections of the GABA agonist muscimol (100 ng/l microliters) into the peripeduncular nucleus. Only the number of tongue protrusions was significantly attenuated, but not that of the ear and cheek movements. Furthermore, tongue protrusions, but no additional oral movements, were elicited by picrotoxin injections (375-500 ng) into the peripeduncular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Origin of the noradrenergic innervation of the superior olivary complex in the rat.

In the rat, the superior olivary complex contains lateral and medial olivocochlear neurones, which respectively innervate two separate targets within the cochlea; the auditory afferents contacting the inner hair cells and the outer hair cells themselves. Previous double label immunohistochemical studies have shown that both lateral and medial olivocochlear neurones are contacted by noradrenergic nerve endings, and electrophysiological studies on in-vitro rat brain slices have demonstrated that noradrenaline exerts a direct, predominantly excitatory effect on medial olivocochlear neurones. In this paper, we have investigated the origin of the noradrenergic input to the superior olivary complex (SOC). A retrograde tracer, Fluorogold, was used to map the inputs to the SOC, and this was combined with immunofluorescent staining for dopamine-beta-hydroxylase (DbetaH) to identify which of the afferent inputs was noradrenergic. These experiments showed small numbers of neurones double-stained for both Fluorogold and DbetaH in the A6 cell group (the locus coeruleus). In the A7 cell group, within and medial to the lateral lemniscus, numerous Fluorogold labelled and DbetaH positive neurones were found, but no neurones were seen that were double-labelled. In none of the other major noradrenergic cell groups were labelled Fluorogold neurones ever detected. To confirm the results obtained by retrograde tracer injections, anterograde tracer injections with biotinylated dextran amine were made in the locus coeruleus. This resulted in labelled fibres within all subdivisions of the superior olivary complex. These experiments indicate that the noradrenergic input to the olivocochlear neurones originates solely from the locus coeruleus. The small numbers of double-labelled neurones found in the locus coeruleus indicate a very divergent non-selective noradrenergic input to the SOC.

Distribution and possible functional roles of some neuroactive peptides in the mammalian superior olivary complex.

The mammalian superior olivary complex (SOC) is innervated by neuronal systems that contain a variety of neuroactive peptides. Conversely, neurones of the SOC form peptidergic projections to other targets. In this review, the peptides substance P, calcitonin-gene-related peptide, enkephalins and dynorphins, cholecystokinin and somatostatin are considered. Their distribution in fibres and cell bodies of the SOC are considered, with particular attention to differences between the SOC subdivisions. Evidence for the functional effects of these peptides is also reviewed and some brief speculations are offered about their possible functional role in hearing.

The hypothalamic paraventricular nucleus in two types of Wistar rats with different stress responses. I. Morphometric comparison.

The present study evaluates the role of the hypothalamic paraventricular nucleus (PVH) in stress regulation by a morphometric comparison of the vascular, neuronal and synaptic properties of this nucleus in two lines of Wistar rats. It has been previously reported that these two lines of rats, indicated as APO-SUS (apomorphine-susceptible) and APO-UNSUS (apomorphine-unsusceptible) rats on the basis of their reactivity to a subcutaneous injection of apomorphine, display a variety of pharmacological and behavioral differences, including differences in their stress-coping mechanisms (Cools et al., Neuropsychobiology, 28 (1993) 100-105). The results show a similar vascular and neuronal organization of the PVH in both lines, but distinct synaptic differences. The PVH (0.12 mm3 volume with about 15,000 neurons on one side) has an overall vascular density of 5.6%, with significant differences between subdivisions (parvocellular central part: 8.3%, parvocellular dorsal/ventral/posterior part: 4.6-5.3%), which means that vascularity is a useful tool to delineate subdivisions in the parvocellular PVH. The neuronal density of 132 x 10(3)/mm3 as found in the present study is two times higher than reported in a previous study Possible reasons for this discrepancy are extensively discussed. The most significant finding of the present study is the observation that APO-SUS rats have a significantly higher synaptic density (158 x 10(6)/mm3) in the PVH than APO-UNSUS rats (108 x 10(6)/mm3). It is discussed in which way this synaptic difference may be correlated with the different activity of the hypothalamo-pituitary-adrenal axis in both lines of Wistar rats.

The hypothalamic paraventricular nucleus in two types of Wistar rats with different stress responses. II. Differential Fos-expression.

The present study investigates the role of corticotropin-releasing hormone (CRH) neurons in stress regulation by a comparison of stress induced Fos-immunoreactivity and CRH-immunoreactivity in the hypothalamic paraventricular nucleus (PVH) of APO-SUS (apomorphine-susceptible), APO-UNSUS (apomorphine-unsusceptible), normal Wistar and adrenalectomized Wistar (ADX) rats. The first two types represent a good model to study the role of the PVH in stress regulation, since they show different stress responses and a differential synaptic organization of the PVH. After placement on an open field for 15 min all rats showed an increase in the number of Fos-immunoreactive nuclei compared to control handling. Interestingly, open field stress, but not control handling, induces significantly fewer Fos-immunoreactive nuclei in the PVH of APO-SUS rats (1255 +/- 49) compared to APO-UNSUS rats (1832 +/- 201). Experiments with ADX rats revealed that 93% of the CRH-immunoreactive neurons contained a Fos-immunoreactive nucleus, which suggests that the differential Fos-expression in APO-SUS and APO-UNSUS rats represents a differential activation of the CRH neurons. This hypothesis is discussed in relation to reported differences in stress responses, stress-induced ACTH levels and synaptic organization of the PVH.