Loss of hilar somatostatin neurons following tetanus toxin-induced seizures

A loss of inhibitory interneurons has been reported in the hippocampus following seizure activity in various animal models of epilepsy and in human epileptic tissue. The question of whether particular populations of inhibitory neurons are similarly affected by the chronic block of inhibition tha tresults after tetanus toxin injections directly into the brain has not previously been addressed. In the present study a unilateral intrahippocampal injection of tetanus toxin into the ventral hippocampus was used to produce a chronic epileptic syndrome characterised by brief seizures that recurred intermittently for 6–8 weeks. The results reveal, for the first time, the morphological changes in somatostatin interneurons following tetanus toxin-induced seizures in the rat. A bilateral short-term increase in immunoreactivity of somatostatin neurons is present 1 week after injection. This is accompanied by an increased intensity of somatostatin-immunoreactive axon terminals in the outer molecular layer of the dentate gyrus, which is more marked on the contralateral side. A chronic and significant loss of somatostatin-immunoreactive neurons was noted in the hilus of the dentate gyrus 2 months later. The significance of the chronic loss of the hilar somatostatin neurons in the control of excitatory activity in the dentate gyrus and whether the acute morphological changes are due to a direct action of the toxin on release mechanisms or as a result of seizure activity are discussed.     

Early cerebrovascular and parenchymal events following prenatal exposure to the putative neurotoxin methylazoxymethanol

One of the most common causes of neurological disabilities are malformations of cortical development (MCD). A useful animal model of MCD consists of prenatal exposure to methylazoxymethanol (MAM), resulting in a postnatal phenotype characterized by cytological aberrations reminiscent of human MCD. Although postnatal effects of MAM are likely a consequence of prenatal events, little is known on how the developing brain reacts to MAM. General assumption is the effects of prenatally administered MAM are short lived (24 h) and neuroblast-specific. MAM persisted for several days after exposure in utero in both maternal serum and fetal brain, but at levels lower than predicted by a neurotoxic action. MAM levels and time course were consistent with a different mechanism of indirect neuronal toxicity. The most prominent acute effects of MAM were cortical swelling associated with mild cortical disorganization and neurodegeneration occurring in absence of massive neuronal cell death. Delayed or aborted vasculogenesis was demonstrated by MAM's ability to hinder vessel formation. In vitro, MAM reduced synthesis and release of VEGF by endothelial cells. Decreased expression of VEGF, AQP1, and lectin-B was consistent with a vascular target in prenatal brain. The effects of MAM on cerebral blood vessels persisted postnatally, as indicated by capillary hypodensity in heterotopic areas of adult rat brain. In conclusion, these results show that MAM does not act only as a neurotoxin per se, but may additionally cause a short-lived toxic effect secondary to cerebrovascular dysfunction, possibly due to a direct anti-angiogenic effect of MAM itself.     

Direct and indirect effects on the lateral geniculate nucleus neurons of prenatal exposure to methylazoxymethanol acetate

In this study the morphology of the lateral geniculate nucleus and occipital cortex in rats with methylazoxymethanol acetate (MAM Ac)-induced micrencephaly was examined. The aim was to examine the relative contributions of (a) the direct cytotoxic action of the drug on precursors of dorsal lateral geniculate nucleus (dLGN) neurons in the fetal brain and, (b) the postnatal degeneration of the dLGN following prenatal destruction of target neurons in the occipital cortex, to the final extent of damage to the dLGN. Exposure to MAM Ac on E13 produced severe necrosis in the fetal thalamus and caused a 77% deficit in neuronal numbers in the mature dLGN. Exposure to MAM Ac on E15 did not cause necrosis in the fetal thalamus but when animals exposed at this time were examined at 5 weeks postnatal age there was an 87% deficit in neuronal numbers in the dLGN. The hypothesis that this deficit was the result of postnatal death of the dLGN neurons following the destruction by MAM Ac of their normal target population in laminae iii and iv of the occipital cortex was supported by the observation of severe postnatal degeneration in the dLGN of animals exposed to MAM Ac on E15. The significance of these direct and indirect effects of the cytotoxic teratogen, MAM Ac, for understanding the mechanisms by which brain abnormalities in human micrencephaly are produced is discussed.     

Absence of striatal newborn neurons with mature phenotype following defined striatal and cortical excitotoxic brain injuries

Experimental stroke and excitotoxic brain lesion to the striatum increase the proliferation of cells residing within the ventricular wall and cause subsequent migration of newborn neuroblasts into the lesioned brain parenchyma. In this study, we clarify the different events of neurogenesis following striatal or cortical excitotoxic brain lesions in adult rats. Newborn cells were labeled by intraperitoneal injection of bromo–deoxy–uridine (BrdU), or by green fluorescent protein (GFP)-expressing lentiviral vectors injected into the subventricular zone (SVZ). We show that only neural progenitors born the first 5 days in the SVZ reside and expand within this neurogenic niche over time, and that these early labeled cells are more prone to migrate towards the striatum as neuroblasts. However, these neuroblasts could not mature into NeuN⁺ neurons in the striatum. Furthermore, we found that cortical lesions, close or distant from the SVZ, could not upregulate SVZ cell proliferation nor promote neurogenesis. Our study demonstrates that both the time window for labeling proliferating cells and the site of lesion are crucial when assessing neurogenesis following brain injury.     

Investigation of striatal dopamine D2 receptor acquisition following prenatal neuroleptic exposure.

To test the hypothesis that neuroleptic blockade impairs the development of striatal dopamine D2 receptors, pregnant rats were given haloperidol, thiothixene, or trifluoperazine for gestational days 15-18 (short-term exposure) or days 5-20 (long-term exposure). All of the drugs were demonstrated to cross the placenta and enter the fetal brains equally well. Striatal dopamine D2 receptors of the pups were assayed on postnatal day 14. Neither receptor density nor receptor affinity was altered significantly by the short- or long-term prenatal neuroleptic treatment.     

Differential sparing of somatostatin-neuropeptide Y and cholinergic neurons following striatal excitotoxin lesions

We previously found that quinolinic acid striatal excitotoxin lesions result in a relative sparing of somatostatin and neuropeptide Y neurons. In the present study we examined dose-response effects of excitotoxins acting at the three subtypes of glutamate receptors: N-methyl-D-aspartate (AA1), quisqualate (AA2), and kainic acid (AA3). Concentrations of both somatostatin-like immunoreactivity (SLI) and neuropeptide a Y-like immunoreactivity (NPYLI) were compared with those of substance P-like immunoreactivity (SPLI) and GABA. Kainic acid (AA3), quisqualic acid (AA2), and AMPA (AA2) resulted in dose-dependent reductions in all four neurochemical markers examined, while N-methyl-D,L-aspartate (AA1) and quinolinic acid (AA1) resulted in relative sparing of SLI and NPYLI. At doses of each excitotoxin which resulted in comparable 50% reductions in both GABA and SPLI only N-methyl-D,L-aspartate and quinolinic acid had no significant effect on concentrations of SLI and NPYLI. The relative sparing of somatostatin-neuropeptide Y neurons was confirmed histologically by using histochemical staining for NADPH-diaphorase neurons combined with either Nissl stains, or immunohistochemical staining for enkephalin. Lesions with N-methyl-D-aspartate agonists resulted in preferential sparing of NADPH-diaphorase neurons while these neurons were more vulnerable than other neurons to kainic acid or AMPA. Choline acetyltransferase neurons were relatively spared, as compared with other neurons, by agents acting at all three glutamate receptor subtypes. N-methyl-D,L-aspartate lesions were blocked with MK-801, while there was no effect on quisqualic acid or kainic acid lesions. The relative sparing of somatostatin-neuropeptide Y neurons following striatal excitotoxin lesions with N-methyl-D-aspartate (AA1) agonists probably reflects a paucity of AA1 receptors on these neurons. Since these neurons are also spared in Huntington's disease, excitotoxins acting at the N-methyl-D-aspartate (AA1) site provide an improved neurochemical model of this illness.     

Sleep-waking cycle in microencephalic rats induced by prenatal methylazoxymethanol application

Sleep-wakefulness cycle of adult microencephalic rats due to prenatal administration of methylazoxymethanol (MAM) acetate at the dose of 20 mg/kg on the 15th day of gestation showed no difference from the normal control group, whereas at the dose of 25 mg/kg, %PS and PS/TS decreased significantly. MAM-induced microencephalic rats revealed a significant increase of spindle bursts during PS, suggesting that their PS is less deep than that of the controls.     

Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat

Previously we described the extent of sprouting that axons of the rat substantia nigra pars compacta (SNpc) undergo to grow new synapses and re-innervate the dorsal striatum 16 weeks after partial lesions. Here we provide insights into the timing of events related to the re-innervation of the dorsal striatum by regenerating dopaminergic nigrostriatal axons over a 104-week period after partial SNpc lesioning. Density of dopamine transporter and tyrosine hydroxylase immunoreactive axonal varicosities (terminals) decreased up to 80% 4 weeks after lesioning but returned to normal by 16 weeks, unless SNpc lesions were greater than 75%. Neuronal tracer injections into the SNpc revealed a 119% increase in axon fibres (4 mm rostral to the SNpc) along the medial forebrain bundle 4 weeks after lesioning. SNpc cells underwent phenotypic changes. Four weeks after lesioning the proportion of SNpc neurons that expressed tyrosine hydroxylase fell from 90% to 38% but returned to 78% by 32 weeks. We discuss these phenotype changes in the context of neurogenesis. Significant reductions in dopamine levels in rats with medium (30-75%) lesions returned to normal by 16 weeks whereas recovery was not observed if lesions were larger than 75%. Finally, rotational behaviour of animals in response to amphetamine was examined. The clear rightward turning bias observed after 2 weeks recovered by 16 weeks in animals with medium (30-75%) lesions but was still present when lesions were larger. These studies provide insights into the processes that regulate sprouting responses in the central nervous system following injury.     

Monoamine neurotransmitter metabolism in microencephalic rat brain after prenatal methylazoxymethanol treatment

Administration of methylazoxymethanol (MAM) in the fetal stage leads to forebrain microencephaly with a severe atrophy in cerebral cortex, striatum, and hippocampus. The concentration of endogenous monoamines was markedly increased in the atrophic regions while total amount was largely unchanged. Striatal dopamine and cortical noradrenaline nerve terminals from MAM treated animals showed unaltered sedimentation properties in a sucrose density gradient and were estimated to have normal transmitter levels. gamma-Butyrolactone induced increase in dopamine levels and its counteraction by apomorphine was essentially unaltered after MAM. These data give further support for the view that the monoamine nerve terminal fields develop to their normal size in the atrophic regions leading to a hyperinnervation. Analysis of monoamine metabolite levels, increase of monoamines after monoamine oxidase inhibition, and disappearance of catecholamines after tyrosine hydroxylase inhibition were conducted to obtain information on monoamine turnover. The results indicated an essentially unaltered, or a small reduction of, monoamine turnover in the atrophic regions when calculated per monoamine nerve terminal, while increased when calculated per unit weight of the tissue.     

Impaired brain development in the rat following prenatal exposure to methylazoxymethanol acetate at gestational day 17 and neurotrophin distribution

Several neuropsychiatric disorders, including schizophrenia, are the consequence of a disrupted development of the CNS. Accordingly, intrauterine exposure to toxins may increase the risk for psychopathology. We investigated whether prenatal exposure of rats to the neurotoxin methylazoxymethanol acetate led to long-term changes in cerebral neurotrophin levels. We measured the brain levels of nerve growth factor and brain derived neurotrophic factor in young adult and adult rats. Decreased nerve growth factor or brain derived neurotrophic factor were found in the parietal cortex accompanied by altered neurotrophin content in the hippocampus and entorhinal cortex. The present study is the first to show long-lasting effects of a single prenatal exposure to a neurotoxin on adult levels of neurotrophins in brain regions implicated in neuropsychiatric disorders.     

Depletion of striatal somatostatin by local cysteamine injection

Local injection of cysteamine into rat striatum resulted in a dose-dependent reduction in somatostatin-like immunoreactivity (SLI). The effect was seen at 1 h, and persisted for up to 72 h, but was reversible at 1 week. The maximal depletion of SLI was approximately 50%. Histologic damage was largely confined to the needle tract. Of interest was a depletion of SLI in the contralateral striatum beginning at 3 h and maximal at 72 h. Cysteamine induced depletion of striatal SLI was not accompanied by alterations in dopamine or serotonin metabolism. Cysteamine is a useful pharmacologic tool for local somatostatin depletion.     

Reduced density of striatal somatostatin receptors in Huntington's chorea

A marked increase of the endogenous somatostatin has been reported in the striatum in Huntington's chorea by radioimmunoassay and immunohistochemistry. Using quantitative receptor autoradiography we examined the density and distribution of somatostatin receptors in the striatum of 6 patients dying from Huntington's chorea degree 3, in 12 control healthy patients dying without neurological diseases and 7 schizophrenic patients, using the stable somatostatin octapeptide analogue [¹²⁵I]204-090 as a radioligand. Marked reductions of the density of somatostatin binding sites were observed in the caudate and putamen of all patients with Huntington's chorea. However, these receptors were well preserved in the nucleus accumbens and in the ventral aspects of the anterior putamen. No alteration of somatostatin receptors was observed in other brain areas. These results suggest that somatostatin receptors in the human striatum are markedly down-regulated or localized on a population of neurons which is at risk in Huntington's chorea and questions the postulated role for the elevated somatostatin levels in choreiform movements.     

Electrophysiological characterization of rat striatal neurons in vitro following a unilateral lesion of dopamine cells

The effects of a unilateral 6 to 19-week lesion of dopamine cells on the excitability of rat striatal neurons were investigated in vitro using the intracellularly recorded membrane properties of neurons obtained ipsilateral and contralateral to 6-hydroxydopamine (6-OHDA) injection sites. Neurons ipsilateral to the lesion site and in striatal tissue depleted of dopamine exhibited resting membrane potentials and membrane resistances similar to those recorded in contralateral striatal neurons. Denervation appeared to have no appreciable effect on the proportion of neurons exhibiting various patterns of neuronal spiking (repetitive, bursting, or single spike) evoked by depolarizing current pulses. Current-voltage determinations revealed nominal rectification in the majority of neurons and marked nonlinearty consistent with inward rectification at potentials hyperpolarized and depolarized to rest in a large proportion of the remaining neurons. Neurons ipsilateral to 6-OHDA lesion sites exhibited these relationships in the same proportion as contralateral control cells. However, ipsilateral neurons with nominal rectification exhibited an average rate constant for the early onset of small hyperpolarizing membrane transients which was significantly smaller than that of controls. This finding suggests that intrinsic membrane parameters regulating the excitability of certain striatal neurons may be under the influence of dopamine or other factors closely associated with nigrostriatal nerve terminals. Published 1993 Wiley-Liss, Inc.     

Striatal depth EEG reveals postsynaptic activity of striatal neurons following dopamine receptor stimulation and blockade

This paper demonstrates a technique for measuring depth electroencephalographic (EEG) recordings from the freely moving mouse. This technique minimizes electrical artifact associated with gross movements by amplifying the current of the EEG signal directly at permanently indwelling electrodes. Stable EEG signals, with high signal-to-noise ratios, can be obtained from these animals while their movement inside the testing cage remains relatively unrestricted. We used this technique to examine the effects of dopamine (DA) receptor agonist and antagonist treatments on depth EEG signals generated within the striatum. Baseline measures of spontaneous striatal EEG activity were obtained prior to drug administration and post-drug measures of striatal activity were subsequently obtained. Apomorphine treatment resulted in desynchronization of striatal EEG signals while haloperidol or sulpiride treatment induced slow wave synchronization. Fast Fourier analysis of EEG signals revealed that DA agonist and antagonist treatment altered spontaneous striatal EEG activity in an opposite manner--relative to baseline signals, apomorphine attenuated low frequency components and augmented higher frequency components of the signal while haloperidol augmented low frequency components and attenuated higher frequency components of the signal. Moreover, mice pretreated with unilateral intracerebral injections of sulpiride and subsequently administered systemic apomorphine simultaneously demonstrated EEG synchronization on the side ipsilateral to the injection of sulpiride and EEG desynchronization on the contralateral side. The population of neurons examined in the medial striatum appear to have the properties of being excitatory to DA agonist stimulation and show decreased activity following DA antagonist treatment. These results suggest that striatal EEG activity may be used as measure of postsynaptic activity of dopaminergic neurons.     

Selective ablation of neurons by methylazoxymethanol during pre- and postnatal brain development

Neonatal or pregnant albino rats were injected with either single or double doses of methylazoxymethanol (20 mg/kg) to test the temporal specificity of its effect on clearly definable regions of the brain. A single dose, to dams from gestational day 11 to 21 (G11-G21) and to neonatal rats from birth to postnatal day 5 (P0-P5), produced differential weight reductions among various brain regions. Two prominent peaks of reduction were found: one occurring between G13 and G15 for the cerebrum and hippocampus and one occurring between P0 and P1 for the cerebellum and olfactory bulbs. Dual injections of the drug on G14 and G15 produced 60% weight reduction in the cerebrum, and slightly earlier injections on G13 and G14 reduced the weight of the cerebellum by about 23%. This weight reduction was accompanied by narrowing of the cerebellar width, which we believe was due to fewer Purkinje cells. Dual injections of methylazoxymethanol at P0 and P1 reduced the weight of the olfactory bulb by 65%, the cerebellum by 62%, and the hippocampus by 18%. These results show that its short action is within the window of cell division for various neurons and becomes additive on two successive days. This precise toxic effect on brain development can be used to disproportionally reduce the number of neuroblasts in specific regions of the brain. A differential ablation allows analysis of plasticity on pyramidal and nonpyramidal cells of the neocortex and hippocampus, Purkinje and granule cells of the cerebellum, and the granule and mitral cells of the olfactory bulbs.     

Opioidergic interactions between striatal projection neurons

Medium spiny striatal projection neurons (MSNs) release opioid neuropeptides, but the role of these neurotransmitters is still poorly understood. While presynaptic inhibition of corticostriatal axons by opioid receptors has been demonstrated using exogenous ligands, the action of synaptically released opioids in the striatum has not been investigated. We performed single and paired whole-cell recordings from rat MSNs while corticostriatal fibers were electrically activated. In single recording experiments, we also activated antidromically the axons of a population of MSNs. Corticostriatal fibers were stimulated once every 10 s and every other stimulation was preceded by 5 antidromic spikes (at 100 Hz). This burst of antidromic spikes produced robust inhibition of evoked corticostriatal responses. This inhibition was not affected by the δ-opioid receptor antagonist SDM25N, but was completely abolished by the μ-opioid receptor antagonist CTOP. Inhibitory effects were maximal (on average 29.6 ± 11.4%) when the burst preceded the corticostriatal stimulation by 500 ms and became undetectable for intervals >2 s. Paired recordings from MSNs located <100 μm apart revealed that, in 30 of 56 (54%) pairs, a burst of five action potentials in one of the MSNs caused significant inhibition (17.1 ± 5.7%) of evoked glutamatergic responses in the other MSN. In 5 of these pairs, reciprocal inhibition of corticostriatal inputs was present. These effects were maximal 500 ms after the burst and were completely blocked by CTOP. Thus, these results reveal a novel, strong opioid-mediated communication between MSNs and provide a new cellular substrate for competitive dynamics in the striatum.     

Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction

Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induction of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale staining in these regions, it is proposed that these islands represent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and infarction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons.     

Sprouting of striatal serotonin nerve terminals following selective lesions of nigro-striatal dopamine neurons in neonatal rat

The effects of neonatal intracisternal 6-hydroxydopamine (6-OHDA; 50 micrograms) treatment on striatal serotonin (5-HT) nerve terminals in rat have been characterized using histo- and neurochemical methods. The 6-OHDA lesion caused a 60% reduction of striatal dopamine (DA) concentration when analyzed in the adult stage, while 5-HT levels were increased by about 40% and 3H-5-HT uptake in vitro was increased by about 60%. Using computerized image analysis, a marked increase in 5-HT-like immunoreactive terminal density was found in both rostral (+200%) and caudal (+50%) striatum. Pretreatment with the DA uptake blocker amfolenic acid completely counteracted the 6-OHDA-induced alterations in both DA and 5-HT neurons in the striatum, while pretreatment with the noradrenaline uptake blocker desipramine had no significant effects. Regional analysis of 5-HT levels in the CNS after neonatal 6-OHDA treatment or the combined desipramine + 6-OHDA treatment showed no significant effect in any of the brain areas analyzed, apart from the observed 5-HT increase in striatum. It was furthermore observed that the striatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio was decreased, while the 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio was increased following the 6-OHDA lesion, indicating compensatory mechanisms in turnover of transmitters. These alterations were completely reversed after pretreatment with amfolenic acid. The present results support the view that the 5-HT hyperinnervation following neonatal 6-OHDA treatment is a collateral sprouting response induced by lesioning of the striatal DA neurons.     

Ascorbate modulates glutamate-induced excitations of striatal neurons

To assess the role of ascorbate (AA), an antioxidant vitamin, in modulating striatal activity, single-unit recording was combined with iontophoresis in awake, unrestrained rats. Brief applications of AA (20 s, 5–80 nA) elicited few changes in either basal activity or activity evoked by continuous application of glutamate (GLU), but relatively high AA ejection currents (>40 nA) often inhibited fast-firing units. Comparable results were obtained with the antioxidant isomer, iso-AA, suggesting the AA-induced inhibition represents a high-dose, antioxidant effect. When applied for prolonged periods (2–4 min) at doses that failed to alter basal activity, AA either enhanced or attenuated the excitatory response to test pulses of GLU. The AA-induced enhancement occurred more frequently (16 vs. 6 applications) and was characterized by a more rapid (shorter onset and peak latencies) and more pronounced (greater peak magnitude) excitation to GLU without an evident change in offset latency. In most cases, further increases in AA ejection current attenuated the GLU response. Iso-AA, in contrast, had only inhibitory effects, which occurred at moderate- to high-dose applications. Collectively, these results suggest that AA, apart from its antioxidant effects, modulates phasic changes in striatal excitability induced by GLU. Because extracellular levels of striatal AA fluctuate in relation to behavioral activation, this neuromodulatory action of AA may contribute to behaviorally relevant changes in sensorimotor responsivity.     

Electrophysiology of ventromedial striatal neurons during movement

The firing rate of ventromedial striatal cons was studied in rats trained to run in place on a rotating turntable treadmill. Animals were trained to run clockwise and counter-clockwise as they propelled a turntable for a water reward. After a period of training, Parylene C coated stainless steel wire electrodes were chronically implanted for single unit recording. Nearly all ventromedial striatal cells increased their firing rate concomitant with locomotion in the treadmill (32 of 36 cells). The magnitude of this response was influenced by the order of testing and by the direction of circling relative to the side of brain being recorded. The increase in firing rate was greater during the first test of the day and was greater when animals circled contralateral to the side of the recording electrode than when they circled ipsilateral to the recorded side.