Orlando Castellano

Origin and function of short-latency inputs to the neural substrates underlying the acoustic startle reflex

The acoustic startle reflex (ASR) is a survival mechanism of alarm, which rapidly alerts the organism to a sudden loud auditory stimulus. In rats, the primary ASR circuit encompasses three serially connected structures: cochlear root neurons (CRNs), neurons in the caudal pontine reticular nucleus (PnC), and motoneurons in the medulla and spinal cord. It is well-established that both CRNs and PnC neurons receive short-latency auditory inputs to mediate the ASR. Here, we investigated the anatomical origin and functional role of these inputs using a multidisciplinary approach that combines morphological, electrophysiological and behavioral techniques. Anterograde tracer injections into the cochlea suggest that CRNs somata and dendrites receive inputs depending, respectively, on their basal or apical cochlear origin. Confocal colocalization experiments demonstrated that these cochlear inputs are immunopositive for the vesicular glutamate transporter 1 (VGLUT1). Using extracellular recordings in vivo followed by subsequent tracer injections, we investigated the response of PnC neurons after contra-, ipsi-, and bilateral acoustic stimulation and identified the source of their auditory afferents. Our results showed that the binaural firing rate of PnC neurons was higher than the monaural, exhibiting higher spike discharges with contralateral than ipsilateral acoustic stimulations. Our histological analysis confirmed the CRNs as the principal source of short-latency acoustic inputs, and indicated that other areas of the cochlear nucleus complex are not likely to innervate PnC. Behaviorally, we observed a strong reduction of ASR amplitude in monaural earplugged rats that corresponds with the binaural summation process shown in our electrophysiological findings. Our study contributes to understand better the role of neuronal mechanisms in auditory alerting behaviors and provides strong evidence that the CRNs-PnC pathway mediates fast neurotransmission and binaural summation of the ASR.

Chronic administration of risperidone to healthy rats: A behavioural and morphological study

Taking into account that most of the experimental research into the effects of antipsychotic drugs has mainly focused on behavioural aspects, the aim of the present work is to investigate the effects of a chronic therapeutic dose of risperidone (1 mg/kg/day during 140 days) on both behavioural and morphological aspects in healthy rats. The behavioural results revealed only minor modifications in prepulse inhibition, showing the risperidone-treated group higher values at 70 days of treatment with respect to the vehicle group. Moreover, in the open-field test, this group showed a greater incidence of grooming. In the active avoidance test, no differences were found between the groups studied. Additionally, in the morphological study performed to analyse cortical thickness and the number of GFAP-, CaBP-, PV- and Fos-immunostained cells no differences were seen between the two groups studied. It is important to note that the risperidone-treated group showed a slight increase in the total number of cells counted, although this increase was not significant. Our results indicate that the chronic administration of therapeutic doses of risperidone does not produce any dramatic behavioural or morphological changes in healthy animals.

The effects of sertraline administration from adolescence to adulthood on physiological and emotional development in prenatally stressed rats of both sexes

Sertraline (SERT) is a clinically effective Selective Serotonin Reuptake Inhibitor (SSRI) known to increase and stabilize serotonin levels. This neurotransmitter plays an important role in adolescent brain development in both rodents and humans, and its dysregulation has been correlated with deficits in behavior and emotional regulation. Since prenatal stress may disturb serotoninergic homeostasis, the aim of this study was to examine the long-lasting effects of exposure to SERT throughout adolescence on behavioral and physiological developmental parameters in prenatally stressed Wistar rats. SERT was administered (5 mg/kg/day p.o.) from the age of 1–3 months to half of the progeny, of both sexes, of gestating dams stressed by use of a restraint (PS) or not stressed. Our data reveal that long-term SERT treatment slightly reduced weight gain in both sexes, but reversed the developmental disturbed “catch-up” growth found in PS females. Neither prenatal stress nor SERT treatment induced remarkable alterations in behavior and had no effects on mean startle reflex values. However, a sex-dependent effects of PS was found: in males the PS paradigm slightly increased anxiety-like behavior in the open field, while in females, it impaired startle habituation. In both cases, SERT treatment reversed the phenomena. Additionally, the PS animals exhibited a disturbed leukocyte profile in both sexes, which was reversed by SERT. The present findings are evidence that continuous SERT administration from adolescence through adulthood is safe in rodents and lessens the impact of prenatal stress in rats.

A fast cholinergic modulation of the primary acoustic startle circuit in rats

Cochlear root neurons (CRNs) are the first brainstem neurons which initiate and participate in the full expression of the acoustic startle reflex. Although it has been suggested that a cholinergic pathway from the ventral nucleus of the trapezoid body (VNTB) conveys auditory prepulses to the CRNs, the neuronal origin of the VNTB–CRNs projection and the role it may play in the cochlear root nucleus remain uncertain. To determine the VNTB neuronal type which projects to CRNs, we performed tract-tracing experiments combined with mechanical lesions, and morphometric analyses. Our results indicate that a subpopulation of non-olivocochlear neurons projects directly and bilaterally to CRNs via the trapezoid body. We also performed a gene expression analysis of muscarinic and nicotinic receptors which indicates that CRNs contain a cholinergic receptor profile sufficient to mediate the modulation of CRN responses. Consequently, we investigated the effects of auditory prepulses on the neuronal activity of CRNs using extracellular recordings in vivo. Our results show that CRN responses are strongly inhibited by auditory prepulses. Unlike other neurons of the cochlear nucleus, the CRNs exhibited inhibition that depended on parameters of the auditory prepulse such as intensity and interstimulus interval, showing their strongest inhibition at short interstimulus intervals. In sum, our study supports the idea that CRNs are involved in the auditory prepulse inhibition of the acoustic startle reflex, and confirms the existence of multiple cholinergic pathways that modulate the primary acoustic startle circuit.

Auditory Prepulse Inhibition of Neuronal Activity in the Rat Cochlear Root Nucleus

Cochlear root neurons (CRNs) provide short-latency acoustic inputs to the caudal pontine reticular nucleus that elicit an acoustic startle reflex (ASR). Auditory prepulse inhibition (PPI) of the acoustic startle response is the reduction in ASR magnitude that is observed when a strong acoustic startling stimulus (pulse) is shortly preceded by a weak sound (prepulse). It has been suggested that a short descending auditory pathway conveys auditory prepulses to the level of the CRNs to mediate the inhibition of ASR. However, no electrophysiological data is available to confirm such inhibition. We here investigated the effects of auditory prepulses on the neuronal activity of CRNs using extracellular recordings in vivo from single CRNs. Our results show that CRN responses are strongly inhibited by auditory prepulses. As occurs in the behavioral paradigm, the inhibition of the CRN responses depended on parameters of the auditory prepulse, such as intensity and interstimulus interval, showing their strongest inhibition at high intensity level and short interstimulus intervals. Furthermore, we tested the auditory PPI on the activity of different neuron types in the ventral cochlear nucleus. Of all neuron types tested, only CRNs exhibited a strong attenuation of activity. Our results corroborate our previous hypothesis that CRNs might be involved in the neural circuit of the inhibition of the ASR and suggest that several neuronal pathways participate in that circuit.

Chronic administration of risperidone in a rat model of schizophrenia: A behavioural, morphological and molecular study

In the present work we analyzed the effect of the chronic administration of risperidone (2mg/kg over 65 days) on behavioural, morphological and molecular aspects in an experimental model of schizophrenia obtained by bilateral injection of ibotenic acid into the ventral hippocampus of new-born rats. Our results show that during their adult lives the animals with hippocampal lesions exhibit different alterations, mainly at behavioural level and in the gene expression of dopamine D(2) and 5-HT(2A) receptors. However, at morphological level the study performed on the prefrontal cortex did not reveal any alterations in either the thickness or the number of cells immunoreactive for c-Fos, GFAP, CBP or PV. Overall, risperidone administration elicited a trend towards the recovery of the values previously altered by the hippocampal lesion, approaching the values seen in the animals without lesions. It may be concluded that the administration of risperidone in the schizophrenia model employed helps to improve the altered functions, with no significant negative effects.

Long-Term Sertraline Intake Reverses the Behavioral Changes Induced by Prenatal Stress in Rats in a Sex-Dependent Way

Early life stress is a major factor underlying the vulnerability to respond to stressful events later in life. The present study attempted to evaluate the role of prenatal stress affecting the development of stress-related disorders and their reversion by postnatal exposure to Sertraline (SERT), a front-line medication for medication for posttraumatic stress disorder (PTSD) in humans. To achieve this, adult male and female prenatally stressed (PS) or unstressed (Controls) offspring rats, following oral chronic treatment with SERT (5 mg/kg/day; from 1 month to 4 months old), or not, were studied prior to and after a traumatic event. First, anxiety-like behavior during the prepulse inhibition (PPI) test, a modulation of the startle reflex, was examined in all animals. Subsequently, the animals were subjected to a session of mild inescapable footshocks (IS; 0.35 mA, 5 s) in a shuttle box that was followed by 4 days of situational reminders in the aversive context. Prior to the footshocks no effects of PS or SERT were shown, and no changes in PPI and the habituation to the shuttle box were found. After them, PS led animals to exhibit behavioral alterations. When compared to the Controls, PS animals of both sexes displayed less rearing activity in the aversive environment. PS males responded less to footshock delivery and, in most of the animals, fear extinction was impaired. Moreover, the early postnatal exposure to SERT lessened the behavioral impact of PS in females, while in males it had no effect. Current results extend previous data from our laboratory, showing that PS heightened vulnerability to stress later on, and that SERT acts differently in males and females.

Direct and indirect nigrofugal projections to the nucleus reticularis pontis caudalis mediate in the motor execution of the acoustic startle reflex

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.