The Left-Right Side-Specific Neuroendocrine Signaling from Injured Brain: An Organizational Principle.

A neurological dogma is that the contralateral effects of brain injury are set through crossed descending neural tracts. We have recently identified a novel topographic neuroendocrine system (T-NES) that operates via a humoral pathway and mediates the left-right side-specific effects of unilateral brain lesions. In rats with completely transected thoracic spinal cords, unilateral injury to the sensorimotor cortex produced contralateral hindlimb flexion, a proxy for neurological deficit. Here, we investigated in acute experiments whether T-NES consists of left and right counterparts and whether they differ in neural and molecular mechanisms. We demonstrated that left- and right-sided hormonal signaling is differentially blocked by the δ-, κ- and µ-opioid antagonists. Left and right neurohormonal signaling differed in targeting the afferent spinal mechanisms. Bilateral deafferentation of the lumbar spinal cord abolished the hormone-mediated effects of the left-brain injury but not the right-sided lesion. The sympathetic nervous system was ruled out as a brain-to-spinal cord-signaling pathway since hindlimb responses were induced in rats with cervical spinal cord transections that were rostral to the preganglionic sympathetic neurons. Analysis of gene-gene co-expression patterns identified the left- and right-side-specific gene co-expression networks that were coordinated via the humoral pathway across the hypothalamus and lumbar spinal cord. The coordination was ipsilateral and disrupted by brain injury. These findings suggest that T-NES is bipartite and that its left and right counterparts contribute to contralateral neurological deficits through distinct neural mechanisms, and may enable ipsilateral regulation of molecular and neural processes across distant neural areas along the neuraxis.

Left-Right Side-Specific Neuropeptide Mechanism Mediates Contralateral Responses to a Unilateral Brain Injury.

Neuropeptides are implicated in control of lateralized processes in the brain. A unilateral brain injury (UBI) causes the contralesional sensorimotor deficits. To examine whether opioid neuropeptides mediate UBI induced asymmetric processes we compared effects of opioid antagonists on the contralesional and ipsilesional hindlimb responses to the left-sided and right-sided injury in rats. UBI induced hindlimb postural asymmetry (HL-PA) with the contralesional hindlimb flexion, and activated contralesional withdrawal reflex of extensor digitorum longus (EDL) evoked by electrical stimulation and recorded with EMG technique. No effects on the interossei (Int) and peroneaus longus (PL) were evident. The general opioid antagonist naloxone blocked postural effects, did not change EDL asymmetry while uncovered cryptic asymmetry in the PL and Int reflexes induced by UBI. Thus, the spinal opioid system may either mediate or counteract the injury effects. Strikingly, effects of selective opioid antagonists were the injury side-specific. The µ-antagonist ß-funaltrexamine (FNA) and κ-antagonist nor-binaltorphimine (BNI) reduced postural asymmetry after the right but not left UBI. In contrast, the δ-antagonist naltrindole (NTI) inhibited HL-PA after the left but not right-side brain injury. The opioid gene expression and opioid peptides were lateralized in the lumbar spinal cord, and coordination between expression of the opioid and neuroplasticity-related genes was impaired by UBI that together may underlie the side-specific effects of the antagonists. We suggest that mirror-symmetric neural circuits that mediate effects of left and right brain injury on the contralesional hindlimbs are differentially controlled by the lateralized opioid system.

Unilateral brain injury to pregnant rats induces asymmetric neurological deficits in the offspring.

Effects of environmental factors may be transmitted to the following generation, and cause neuropsychiatric disorders including depression, anxiety, and posttraumatic stress disorder in the offspring. Enhanced synaptic plasticity induced by environmental enrichment may be also transmitted. We here test the hypothesis that the effects of brain injury in pregnant animals may produce neurological deficits in the offspring. Unilateral brain injury (UBI) by ablation of the hindlimb sensorimotor cortex in pregnant rats resulted in the development of hindlimb postural asymmetry (HL-PA), and impairment of balance and coordination in beam walking test in the offspring. The offspring of rats with the left UBI exhibited HL-PA before and after spinal cord transection with the contralesional (i.e., right) hindlimb flexion. The right UBI caused the offspring to develop HL-PA that however was cryptic and not-lateralized; it was evident only after spinalization, and was characterized by similar occurrence of the ipsi- and contralesional hindlimb flexion. The HL-PA persisted after spinalization suggesting that the asymmetry was encoded in lumbar spinal neurocircuits that control hindlimb muscles. Balance and coordination were affected by the right UBI but not the left UBI. Thus, the effects of a unilateral brain lesion in pregnant animals may be intergenerationally transmitted, and this process may depend on the side of brain injury. The results suggest the existence of left-right side-specific mechanisms that mediate transmission of the lateralized effects of brain trauma from mother to fetus.

Altered serotonin innervation in the rat epileptic brain.

Studies in animal models of epilepsy revealed compromised serotonin (5-HT) transmission between the raphe nuclei and the brain limbic system. The goal of the present study was to evaluate the effects of epilepsy on the structural integrity of the dorsal (DR) and median (MnR) raphe nuclei and on the morphology of serotonergic fiber terminals in the dentate gyrus (DG), infralimbic cortex (IL) and medial septum (MS). The study was performed in adult Wistar rats using the kainate (9.5 mg/kg) status epilepticus (SE) model. Four months post-SE, the brainstem sections of the animals were immunostained for 5-HT, whereas the forebrain sections were immunostained for serotonin transporter (SERT). Stereological analysis revealed that epileptic rats, as compared to controls, had approximately 30% less 5-HT-stained cells in the interfascicular part of the DR, but twice as many 5-HT-stained cells in the MnR. Another finding was the reorganization of the 5-HT fiber network in all target areas analyzed, as indicated by the rightward shift of the density-size distribution histograms of SERT-stained fiber varicosities. Nonlinear regression analysis of these histograms revealed that SERT-stained varicosities were represented by two subpopulations characterized by distinct cross-sectional areas. The areal density of the small-sized varicosities was decreased in the DG (hilus and molecular layer), IL cortex (layers II/III) and MS, while that of the larger-sized varicosities was increased. The present results support the hypothesis that chronic epilepsy can trigger profound structural reorganization of the ascending serotonergic pathways in the rat brain.

Partial depletion of septohippocampal cholinergic cells reduces seizure susceptibility, but does not mitigate hippocampal neurodegeneration in the kainate model of epilepsy.

The brain cholinergic system may undergo structural and functional alterations both in human epilepsy and in respective animal models, but the causal relationships between these alterations and epilepsy remain to be established. In this study, we attempted to examine how the inhibition of epilepsy-related cholinergic plasticity may be reflected in seizure susceptibility and/or in the development of chronic epilepsy and its neurological consequences. For this purpose, adult Wistar rats received intrahippocampal injections of low doses of 192-IgG-saporin (SAP) to produce a moderate, but significant loss of septohippocampal cholinergic cells and to suppress their plasticity. Then, animals were treated with kainic acid to induce status epilepticus, which leads to the development of chronic epilepsy later in life. It was found that SAP-pretreatment was associated with longer latency to the onset of status epilepticus and with reduced mortality rate, suggesting that increased activity of septal cholinergic cells may potentiate seizures. Interestingly, months later, a greater percentage of rats with intact septohippocampal cholinergic connections showed spontaneous seizures, when compared to SAP-pretreated rats. Treatment with kainic acid produced death of 40-50% of hippocampal neurons and this effect was not ameliorated by prior cholinergic depletion. Moreover, the kainate induced cognitive deficits were detected in both SAP-pretreated and sham-pretreated groups. These data suggest that seizure-induced plasticity of cholinergic cells may indeed enhance seizure susceptibility and contribute to epileptogenic processes. They do not support the hypothesis that epilepsy-related hypertrophy of cholinergic neurons may potentiate hippocampal cell loss and respective behavioral impairments.

The pedunculopontine and laterodorsal tegmental nuclei in the kainate model of epilepsy.

Prior studies showed that epilepsy can be associated with reorganization of the septohippocampal cholinergic fiber system. Using the kainate model of epilepsy, we wished to further examine the structural integrity of the mesopontine tegmental nuclei (pedunculopontine, PPN, and laterodorsal, LDT), which provide the cholinergic input to the thalamus. It was found that the total numbers of the PPN and LDT cells immunoreactive to the vesicular acetylcholine transporter did not differ between control and epileptic rats. However, the cholinergic cells had enlarged perikarya in epileptic rats. We further examined the effects of epilepsy on the distribution pattern of cholinergic fiber varicosities in the parafascicular nucleus, one of the principal thalamic targets of PPN projections. The density of cholinergic varicosities, represented by two distinct populations, was increased in epileptic rats. These data provide the first morphological evidence for structural alterations in mesopontine cholinergic neurons in experimental epilepsy. They suggest dysfunctional cholinergic transmission in the brainstem-thalamic pathway, which may partly account for various epilepsy-related neurological disturbances.

Serotonin depletion increases seizure susceptibility and worsens neuropathological outcomes in kainate model of epilepsy.

Serotonin is implicated in the regulation of seizures, but whether or not it can potentiate the effects of epileptogenic factors is not fully established. Using the kainic acid model of epilepsy in rats, we tested the effects of serotonin depletion on (1) susceptibility to acute seizures, (2) development of spontaneous recurrent seizures and (3) behavioral and neuroanatomical sequelae of kainic acid treatment. Serotonin was depleted by pretreating rats with p-chlorophenylalanine. In different groups, kainic acid was injected at 3 different doses: 6.5mg/kg, 9.0mg/kg or 12.5mg/kg. A single dose of 6.5mg/kg of kainic acid reliably induced status epilepticus in p-chlorophenylalanine-pretreated rats, but not in saline-pretreated rats. The neuroexcitatory effects of kainic acid in the p-chlorophenylalanine-pretreated rats, but not in saline-pretreated rats, were associated with the presence of tonic-clonic convulsions and high lethality. Compared to controls, a greater portion of serotonin-depleted rats showed spontaneous recurrent seizures after kainic acid injections. Loss of hippocampal neurons and spatial memory deficits associated with kainic acid treatment were exacerbated by prior depletion of serotonin. The present findings are of particular importance because they suggest that low serotonin activity may represent one of the major risk factors for epilepsy and, thus, offer potentially relevant targets for prevention of epileptogenesis.

Reorganization of the septohippocampal cholinergic fiber system in experimental epilepsy.

The septohippocampal cholinergic neurotransmission has long been implicated in seizures, but little is known about the structural features of this projection system in epileptic brain. We evaluated the effects of experimental epilepsy on the areal density of cholinergic terminals (fiber varicosities) in the dentate gyrus. For this purpose, we used two distinct post-status epilepticus rat models, in which epilepsy was induced with injections of either kainic acid or pilocarpine. To visualize the cholinergic fibers, we used brain sections immunostained for the vesicular acetylcholine transporter. It was found that the density of cholinergic fiber varicosities was higher in epileptic rats versus control rats in the inner and outer zones of the dentate molecular layer, but it was reduced in the dentate hilus. We further evaluated the effects of kainate treatment on the total number, density, and soma volume of septal cholinergic cells, which were visualized in brain sections stained for either vesicular acetylcholine transporter or choline acetyltransferase (ChAT). Both the number of septal cells with cholinergic phenotype and their density were increased in epileptic rats when compared to control rats. The septal cells stained for vesicular acetylcholine transporter, but not for ChAT, have enlarged perikarya in epileptic rats. These results revealed previously unknown details of structural reorganization of the septohippocampal cholinergic system in experimental epilepsy, involving fiber sprouting into the dentate molecular layer and a parallel fiber retraction from the dentate hilus. We hypothesize that epilepsy-related neuroplasticity of septohippocampal cholinergic neurons is capable of increasing neuronal excitability of the dentate gyrus.

Altered taste preference and loss of limbic-projecting serotonergic neurons in the dorsal raphe nucleus of chronically epileptic rats.

Mood disorders and major depression are frequently comorbid with epilepsy. While the nature of this comorbidity is not fully understood, multiple lines of evidence suggest that changes in serotonin (5-HT) neurotransmission may be an underlying mechanism. In this study, we tested the hypothesis that chronic epilepsy in rats can be associated with loss of 5-HT neurons in the dorsal raphe (DR) nuclear complex, the main source of 5-HT projections to the cerebral cortex, which would help to explain respective behavioral deficits. Epilepsy was induced using the kainate model of status epilepticus in adult Wistar rats. After a 3-month recovery period, all kainate-treated rats that had experienced status epilepticus showed spontaneous seizures and reduced sucrose preference (anhedonia), a core symptom of depression. No changes in the forced swim test were detected. The total numbers of 5-HT immunoreactive cells were estimated in all DR subdivisions of control and epileptic rats. Interestingly, epilepsy-related loss of 5-HT neurons (approximately 35%) was observed only in the interfascicular part of the DR complex, which is known to innervate brain regions involved in depression. These findings support the notion that mental health impairments observed in epilepsy may be related to loss of a specific population of the DR 5-HT neurons projecting to limbic brain areas.

Loss of hippocampal neurons after kainate treatment correlates with behavioral deficits.

Treating rats with kainic acid induces status epilepticus (SE) and leads to the development of behavioral deficits and spontaneous recurrent seizures later in life. However, in a subset of rats, kainic acid treatment does not induce overt behaviorally obvious acute SE. The goal of this study was to compare the neuroanatomical and behavioral changes induced by kainate in rats that developed convulsive SE to those who did not. Adult male Wistar rats were treated with kainic acid and tested behaviorally 5 months later. Rats that had experienced convulsive SE showed impaired performance on the spatial water maze and passive avoidance tasks, and on the context and tone retention tests following fear conditioning. In addition, they exhibited less anxiety-like behaviors than controls on the open-field and elevated plus-maze tests. Histologically, convulsive SE was associated with marked neuron loss in the hippocampal CA3 and CA1 fields, and in the dentate hilus. Rats that had not experienced convulsive SE after kainate treatment showed less severe, but significant impairments on the spatial water maze and passive avoidance tasks. These rats had fewer neurons than control rats in the dentate hilus, but not in the hippocampal CA3 and CA1 fields. Correlational analyses revealed significant relationships between spatial memory indices of rats and neuronal numbers in the dentate hilus and CA3 pyramidal field. These results show that a part of the animals that do not display intense behavioral seizures (convulsive SE) immediately after an epileptogenic treatment, later in life, they may still have noticeable structural and functional changes in the brain.