Laura Mòdol

Neurosteroids infusion into the CA1 hippocampal region on exploration, anxiety-like behaviour and aversive learning.

Neurosteroids (NS) are substances synthesised de novo in the brain that have rapid modulatory effects on ionotropic receptors. Specifically, NS can act as positive allosteric modulators of GABAA receptors as pregnanolone or allopregnanolone (Allop), or GABAA negative modulators and NMDA positive modulators as pregnenolone (PREG) or dehydroepiandrosterone (DHEA) and their sulphate esters (PREGS and DHEAS). Given this, their role in anxiety and emotional disturbances has been suggested. In addition, NS such as PREGS or DHEAS have demonstrated a promnesic role in several learning tests. The aim of the present work is to highlight the role that the dorsal (CA1) hippocampus plays in the behavioural profile of NS such as Allop and PREGS in tests assessing exploration, anxiety and aversive learning in rats. For this purpose, animals were administered intrahippocampally with Allop (0.2μg/0.5μl), PREGS (5ng/0.5μl) or vehicle in each hippocampus, and tested in the Boissier and elevated plus maze (EPM) tests. For learning test we have chosen the passive avoidance paradigm. Results indicate that intrahippocampal administration of Allop enhances exploration, reflected in an increase in the total and the inner number of head-dips. Allop-injected animals also showed an increase in the percentage of entries into the open arms of the EPM, suggesting an anxiolytic-like profile. In addition, post-acquisition PREGS administration enhanced passive avoidance retention, while post-acquisition Allop administration had no effects on aversive learning retention. These results point out the important role of the dorsal (CA1) hippocampus in several NS behavioural effects, such as exploration, anxiety, learning and memory.

Early increasing-intensity treadmill exercise reduces neuropathic pain by preventing nociceptor collateral sprouting and disruption of chloride cotransporters homeostasis after peripheral nerve injury.

Abstract Activity treatments, such as treadmill exercise, are used to improve functional recovery after nerve injury, parallel to an increase in neurotrophin levels. However, despite their role in neuronal survival and regeneration, neurotrophins may cause neuronal hyperexcitability that triggers neuropathic pain. In this work, we demonstrate that an early increasing-intensity treadmill exercise (iTR), performed during the first week (iTR1) or during the first 2 weeks (iTR2) after section and suture repair of the rat sciatic nerve, significantly reduced the hyperalgesia developing rapidly in the saphenous nerve territory and later in the sciatic nerve territory after regeneration. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in sensory neurons and spinal cord was reduced in parallel. iTR prevented the extension of collateral sprouts of saphenous nociceptive calcitonin gene–related peptide fibers within the adjacent denervated skin and reduced NGF expression in the same skin and in the L3 dorsal root ganglia (DRG). Injury also induced Na+–K+–2Cl− cotransporter 1 (NKCC1) upregulation in DRG, and K+–Cl− cotransporter 2 (KCC2) downregulation in lumbar spinal cord dorsal horn. iTR normalized NKCC1 and boosted KCC2 expression, together with a significant reduction of microgliosis in L3-L5 dorsal horn, and a reduction of BDNF expression in microglia at 1 to 2 weeks postinjury. These data demonstrate that specific activity protocols, such as iTR, can modulate neurotrophins expression after peripheral nerve injury and prevent neuropathic pain by blocking early mechanisms of sensitization such as collateral sprouting and NKCC1/KCC2 disregulation.

Prevention of NKCC1 phosphorylation avoids downregulation of KCC2 in central sensory pathways and reduces neuropathic pain after peripheral nerve injury

Summary Inhibition of NKCC1 activation after peripheral nerve injury prevents decrease of KCC2 in the central pain projecting areas and is associated with reduction of hyperalgesia. ABSTRACT Neuropathic pain after peripheral nerve injury is characterized by loss of inhibition in both peripheral and central pain pathways. In the adult nervous system, the Na+–K+–2Cl− (NKCC1) and neuron‐specific K+–Cl− (KCC2) cotransporters are involved in setting the strength and polarity of GABAergic/glycinergic transmission. After nerve injury, the balance between these cotransporters changes, leading to a decrease in the inhibitory tone. However, the role that NKCC1 and KCC2 play in pain‐processing brain areas is unknown. Our goal was to study the effects of peripheral nerve injury on NKCC1 and KCC2 expression in dorsal root ganglia (DRG), spinal cord, ventral posterolateral (VPL) nucleus of the thalamus, and primary somatosensory (S1) cortex. After sciatic nerve section and suture in adult rats, assessment of mechanical and thermal pain thresholds showed evidence of hyperalgesia during the following 2 months. We also found an increase in NKCC1 expression in the DRG and a downregulation of KCC2 in spinal cord after injury, accompanied by later decrease of KCC2 levels in higher projection areas (VPL and S1) from 2 weeks postinjury, correlating with neuropathic pain signs. Administration of bumetanide (30 mg/kg) during 2 weeks following sciatic nerve lesion prevented the previously observed changes in the spinothalamic tract projecting areas and the appearance of hyperalgesia. In conclusion, the present results indicate that changes in NKCC1 and KCC2 in DRG, spinal cord, and central pain areas may contribute to development of neuropathic pain.

Neonatal neurosteroid levels are determinant in shaping adult prepulse inhibition response to hippocampal allopregnanolone in rats

Diverse studies indicate that the alteration of the physiological levels of neurosteroids in early neonatal phases provokes alterations in the maturation of certain cerebral structures. Allopregnanolone (ALLO) has important modulatory effects in the hippocampus during the postnatal period where the adult pattern of inhibitory transmission is being established. In order to study whether endogenous neonatal ALLO levels would be a determinant parameter involved in mediating adult hippocampal GABAA system maturation, we investigated the effects of neonatal finasteride (50mg/kg, SC) treatment and ALLO (ALLO; 20mg/kg, SC) supplementation on an animal behavioural model with relevance to neurodevelopmental disorder, such as schizophrenia. Two sets of experiments were conducted. Neonatal treatment (from postnatal day (pnd) 5 to pnd9) was performed in 23 male Wistar rats and steroid quantification was performed in hippocampal homogenates at pnd9. A second group (n=127) underwent neonatal treatment (pnd5-pnd9) and were submitted to hippocampal surgery at 80d. The behavioural response to bilateral intrahippocampal neurosteroid administration (ALLO, 0.2μg/0.5μl per side or pregnenolone sulphate 5ng/0.5μl per side) on novelty-induced exploration activity and prepulse inhibition (PPI) was assessed at 95d. Results showed that neonatal ALLO and finasteride administration decreased novelty directed exploratory behaviour and impaired the prepulse inhibition of the acoustic startle response at 95 days of age. Moreover, intrahippocampal ALLO increased head-dipping behaviour independently of the neonatal treatment, while intrahippocampal ALLO decreased PPI only in finasteride and ALLO groups. The results obtained in the present study indicate the importance of neonatal neurosteroid levels in the development of hippocampal function and their relevance in a behavioural phenotype that some have likened to that present in schizophrenia.

Neonatal allopregnanolone levels alteration: Effects on behavior and role of the hippocampus

Several works have pointed out the importance of the neurosteroid allopregnanolone for the maturation of the central nervous system and for adult behavior. The alteration of neonatal allopregnanolone levels in the first weeks of life alters emotional adult behavior and sensory gating processes. Without ruling out brain structures, some of these behavioral alterations seem to be related to a different functioning of the hippocampus in adult age. We focus here on the different behavioral studies that have revealed the importance of neonatal allopregnanolone levels for the adult response to novel environmental stimuli, anxiety-related behaviors and processing of sensory inputs (prepulse inhibition). An increase in neonatal physiological allopregnanolone levels decreases anxiety and increases novelty responses in adult age, thus affecting the individual response to environmental cues. These effects are also accompanied by a decrease in prepulse inhibition, indicating alterations in sensory gating that have been related to that present in disorders, such as schizophrenia. Moreover, behavioral studies have shown that some of these effects are related to a different functioning of the dorsal hippocampus, as the behavioral effects (decrease in anxiety and locomotion or increase in prepulse inhibition) of intrahippocampal allopregnanolone infusions in adult age are not present in those subjects in whom neonatal allopregnanolone levels were altered. Recent data indicated that this hippocampal involvement may be related to alterations in the expression of gamma-aminobutyric-acid receptors containing α4 and δ subunits, molecular alterations that can persist into adult age and that can, in part, explain the reported behavioral disturbances.

Alteration of neonatal Allopregnanolone levels affects exploration, anxiety, aversive learning and adult behavioural response to intrahippocampal neurosteroids

Neurosteroids (NS) are well known to exert modulatory effects on ionotropic receptors. Recent findings indicate that NS could also act as important factors during development. In this sense, neonatal modifications of Allopregnanolone (Allop) levels during critical periods have been demonstrate to alter the morphology of the hippocampus but also other brain structures. The aim of the present work is to screen whether the alterations of Allop levels modify adult CA1 hippocampal response to NS administration. For this purpose, pups were injected with Allop (20 mg/kg s.c.), Finasteride (5α-reductase inhibitor that impedes Allop synthesis) (50 mg/kg s.c.) or Vehicle from postnatal day 5 (P5) to postnatal day 9 (P9). NS levels were tested at P5. To test the behavioural hippocampal response to NS in adulthood, animals were implanted with a bilateral cannula into the CA1 hippocampus at 80 days old and injected with Allop (0.2 μg/0.5 μl), Pregnenolone sulphate (5 ng/0.5 μl) or Vehicle in each hippocampus. After injections animals were tested in the Boisser test to assess exploratory behaviour, the elevated plus maze to assess anxiety and the passive avoidance to test aversive learning. Results indicate that alteration of neonatal Allop or pregnenolone levels (by Allop and Finasteride administration, respectively) suppressed intrahippocampal Allop anxiolytic effect in the EPM. Moreover our results also indicate that manipulation of neonatal Allop levels (Allop and Finast administration) alters exploratory and anxiety-like behaviour and impairs aversive learning in the adulthood. These data point out the role of Allop in the maturation of hippocampal function and behaviour.

Allopregnanolone infused into the dorsal (CA1) hippocampus increases prepulse inhibition of startle response in Wistar rats

The hippocampus is a brain structure that has traditionally been associated with the pathophysiology and neuropathology of schizophrenia. Also, one of the animal models of schizophrenia most widely accepted and validated is the deterioration of prepulse inhibition (PPI). The hippocampus (both dorsal and ventral) seems to be a brain structure important for the PPI since it appears to contribute to sensorimotor gating. A possible role of neurosteroids in schizophrenia has recently been suggested, as clozapine and olanzapine treatments increase brain and plasma levels of the neurosteroid allopregnanolone (AlloP). The aim of the present work is to investigate the effects of the intrahippocampal administration of neurosteroids on prepulse inhibition. For this purpose, we have bilaterally injected AlloP (0.2 μg/0.5 μl) and pregnenolone sulphate (PregS, 5 ng/0.5 μl) into the dorsal CA1 hippocampus, and we have evaluated PPI behavior. Results show that intrahippocampal AlloP increases PPI ability regardless of prepulse intensity (5, 10 or 15 db above background). Intrahippocampal PregS administration, at the dose tested, does not significantly affect PPI performance. The increase in PPI due to intrahippocampal AlloP administration points out the important role of the hippocampus in central sensorimotor gating mechanisms and on the effects of neurosteroids on this behavior. The present findings may contribute to the study of the neurobiological basis of schizophrenia.

Neonatal finasteride administration alters hippocampal α4 and δ GABAAR subunits expression and behavioural responses to progesterone in adult rats

Allopregnanolone is a neurosteroid that has been reported to fluctuate during early developmental stages. Previous experiments reported the importance of neonatal endogenous allopregnanolone levels for the maturation of the central nervous system and particularly for the hippocampus. Changes in neonatal allopregnanolone levels have been related to altered adult behaviour and with psychopathological susceptibility, including anxiety disorders, schizophrenia and drug abuse. However, the mechanism underlying these changes remains to be elucidated. In the present study we assessed changes in hippocampal expression of α4 and δ GABAA receptor (GABAAR) subunits as a consequence of neonatal finasteride (a 5-α reductase inhibitor) administration during early development (PD6 to PD15) in male rats. We observed that the treatment altered the temporal window of the natural peak in the expression of these subunits during development. Additionally, the level of these subunits were higher than in non-handled and control animals in the adult hippocampus. We observed that in adulthood, neonatal finasteride-treated animals presented an anxiogenic-like profile in response to progesterone administration which was absent in the rest of the groups. In conclusion, these results corroborate the relevance of neonatal maintenance of neurosteroid levels for behavioural anxiety responses in the adult, and point to some of the mechanisms involved in this alterations.

Differential effects on KCC2 expression and spasticity of ALS and traumatic injuries to motoneurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease manifested by progressive muscle atrophy and paralysis due to the loss of upper and lower motoneurons (MN). Spasticity appears in ALS patients leading to further disabling consequences. Loss of the inhibitory tone induced by downregulation of the potassium chloride cotransporter 2 (KCC2) in MN has been proposed to importantly contribute to the spastic behavior after spinal cord injury (SCI). The aim of the present study was to test whether the alterations in the expression of KCC2 are linked to the appearance of spasticity in the SODG93A ALS murine model. We compared SODG93A mice to wild type mice subjected to SCI to mimic the spinal MN disconnection from motor descending pathways, and to sciatic nerve lesion to mimic the loss of MN connectivity to muscle. Electrophysiological results show that loss of motor function is observed at presymptomatic stage (8 weeks) in SODG93A mice but hyperreflexia and spasticity do not appear until a late stage (16 weeks). However, KCC2 was not downregulated despite MN suffered disconnection both from muscles and upper MNs. Further experiments revealed decreased gephyrin expression, as a general marker of inhibitory systems, accompanied by a reduction in the number of Renshaw interneurons. Moreover, 5-HT fibers were increased in the ventral horn of the lumbar spinal cord at late stage of disease progression in SOD1G93A mice. Taken together, the present results indicate that spasticity appears late in the ALS model, and may be mediated by a decrease in inhibitory interneurons and an increase of 5-HT transmission, while the absence of down-regulation of KCC2 could rather indicate an inability of MNs to respond to insults.

Interaction between neonatal allopregnanolone administration and early maternal separation: effects on adolescent and adult behaviors in male rat.

Endogenous neurosteroid level fluctuations are related to several emotional and behavioral alterations. Neurosteroids also have important roles during neurodevelopment, with there being a relationship between modification of their levels in neurodevelopmental periods and behavioral alterations in adolescence and adulthood. Early maternal separation (EMS) is a stressful event that also alters neurodevelopment and adolescent and adult behaviors. The aim of the present study is to analyze the interaction between the effects of the neonatal alteration of allopregnanolone (AlloP), neurosteroid that increase its levels after acute stress presentation, and EMS on adolescent exploration and adult anxiety and sensorimotor gating in male rats. AlloP (10 mg/kg s.c.) was administrated between postnatal day 5 (PN5) and PN9, and a single 24-hour period of EMS was carried out on PN9. Exploration was analyzed at PN40 and PN60. At adult age (PN85), anxiety was tested by means of the elevated plus-maze test (EPM), and sensorimotor gating by means of prepulse inhibition test (PPI). PPI deterioration has been considered as a reliable indicator of diseases such as schizophrenia. Results showed that the previous neonatal AlloP administration neutralized the effects of EMS in the adolescent exploration (increase of traveled distance and decrease of head-dips). In adult age, an anxiolytic-like profile was observed as a consequence of EMS. Finally, EMS and neonatal AlloP disrupted PPI. Taken together, these data show the important role that physiological neonatal AlloP levels and stressful events play in neural development, adult behavior and vulnerability to neurodevelopmental disorders such as schizophrenia.