Possible destruction of cholinoceptive neurons by overstimulation of cholinergic tracts

It is well established that intracerebral injections of kainic acid may cause not only neuronal cell destruction at the injection site, but also losses in some distant regions. The mechanisms are different. The distant, but not the local, destruction can be produced by folic as well as by kainic acid and prevented by pretreatment of the animal with diazepam. Overexcitation of excitatory projections is believed responsible for the distant damage and evidence is presented that in some instances the projections involved are cholinergic. Thus, for example, injections of kainic acid or folic acid into the substantia innominata of rats destroy neurons in areas such as the pyriform cortex and amygdala which receive cholinergic projections from the injected area. Some of the destroyed neurons are GABAergic. That the distant toxicity in these areas can be partially blocked by scopolamine and is accompanied by decreases in the number of muscarinic binding sites is consistent with a cholinergic mechanism. Distant damage also occurs in the thalamus but this appears to be mediated by a noncholinergic projection. Similar injections of folic acid or kainic acid into the rostral pontine tegmentum, another area with cholinergic cells, cause destruction of both dopaminergic and GABAergic neurons in the substantia nigra. The effect on the GABAergic but not that on the dopaminergic cells is blocked by scopolamine. The results are discussed in relation to possible mechanisms of epilepsy and of selective neuronal losses in diseases such as Parkinson's disease.     

Some factors influencing the neurotoxicity of intrastriatal injections of kainic acid

Intrastriatal injections of kainic acid are known to destroy striatal neurons including many containing choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD). Using these enzymes as indices of neuronal loss, the neurotoxicity of small doses of kainic acid was found to be influenced by injection time and volume. It was partly blocked by coninjection of some but not all glutamate antagonists or by prior lesioning of the corticostriatal tract. Other adjuvants, drugs, or lesions tested had little modifying effect, except that changes in the dopaminergic system seemed to increase the toxicity towards cholinergic but not GABAnergic systems. High-affinity glutamate accumulation by neostriatal synaptosomes was significantly increased 1–7 days following kainic acid injections. MAO and acetylcholinesterase activities were depressed in kainic acid-lesioned striata but not nearly as much as were CAT and GAD. An indirect mechanism involving glutamate release and inhibition of reuptake is suggested for kainic acid neurotoxicity.     

LOCALIZATION OF GLUTAMINASE IN THE RAT NEOSTRIATUM

Abstract— Glutaminase activity in rat striatal tissue was not significantly decreased by lesions of the cortico-striatal tract which depressed striatal glutamate uptake by 47% but was markedly decreased following intrastriatal injections of kainic acid. There also appeared to be a linear correlation between glutaminase and glutamic acid decarboxylase activities in the substantia nigra of rats injected intrastriatally with kainic acid. The results suggest that most of the glutaminase activity in these regions is localized in GABAergic structures and provide no evidence for the occurrence of this enzyme in nerve endings of the glutamergic cortico-striatal tract.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.     

Effects of zinc sulfate on limbic motor epilepsy induced by kainic acid

In the present study the Authors have investigated the effects of chronic pretreatment with injections of Zn++ sulphate solution on kainic acid induced epilepsy, to verify wether or not Zn++ is able to change the epileptiform pattern induced by kainic acid infection. Results obtained show that Zn++ do not produce any significant change in the experimental parameters by us study.     

Neurochemical sequelae of kainate injections in corpus striatum and substantia nigra of the rat.

Unilateral injection of 2 μg kainic acid into the substantia nigra of the rat results in a 45% decrease in tyrosine hydroxylase activity in the injected substantia nigra and in the ipsilateral corpus striatum. In contrast, the GABAergic nerve terminals in the substantia nigra are unaffected by this treatment. Injection of kainic acid into the striatum results in a 60% decrement in the activity of glutamate decarboxylase and of endogenous GABA levels in the ipsilateral substantia nigra whereas tyrosine hydroxylase activity remains unchanged; in addition, dopamine-sensitive adenylate cyclase activity in the ipsilateral substantia nigra decreases by 74%. These findings further support the hypothesis that intracerebral injections of kainic acid cause degeneration of neurons with cell bodies near the injection site while sparing axons passing through or terminating in the region.     

Effects of phencyclidine on schedule-controlled responding following neurotoxic lesions of the striatum

The effects of phencyclidine on an operant task were evaluated prior to and after neurotoxic lesions of the striatum in rats. Subjects were trained to respond on a fixed-interval 90-second schedule for water presentation. The degree to which phencyclidine disrupted responding was first evaluated (dose range 1.0-4.0 mg/kg). The subjects were then divided into three matched groups and received bilateral intraventricular injections of 6-hydroxydopamine (6-OHDA) (100 microg), kainic acid (0.25 microg), or vehicle delivered stereotaxically. 6-OHDA was used to destroy the presynaptic neurons of the nigro-striatal pathway and kainic acid was employed to destroy the postsynaptic neurons whose cell bodies are located in the striatum. Following recovery, the phencyclidine dose-response curve was repeated in the fixed-interval paradigm. It was observed that 6-OHDA-induced damage resulted in a rightward shift of the dose-response curve indicating tolerance to phencyclidine and caused a significant depletion of striatal dopamine and gamma-aminobutyric acid (GABA). Kainic acid-induced damage resulted in a leftward shift in the dose-response curve indicating sensitivity to the schedule-disruptive effects of phencyclidine and produced a significant GABA depletion. The vehicle-treated rats exhibited no shift in their sensitivity to phencyclidine. These observations indicate that the effects of phencyclidine are mediated, at least in part, by striatal dopaminergic neurons.     

Imaging of the Degeneration of Neurons and Their Processes in Rat or Cat Brain by 45CaCl2 Autoradiography or 55CoCl2 Positron Emission Tomography

The possibility of using radiolabeled divalent cations to visualize nerve cell degeneration in the brain was investigated after intoxication with neurotoxins. At different survival times after the intracerebral injection of kainic acid or 6-hydroxydopamine, autoradiographs were made from brain sections of rats that had received 45CaCl2 intravenously 24 h before death. Brain sections, adjacent to those used for autoradiography, of the 6-hydroxydopamine-treated rats were used for histofluorescence of catecholamines to check the neurochemical effect of the treatment. These experiments show that radioactive Ca accumulates in brain tissue during a particular phase of degeneration. Not only could degenerating cell bodies be traced by 45Ca autoradiography, but also degenerating nerve terminals in the striato-nigral and nigro-striatal projection systems. In positron emission tomography (PET) studies, 55CoCl2 was used as a marker for Ca2+. Unilateral lesions of the cat forebrain, produced by kainic acid, could be imaged in vivo by PET with 55CoCl2. PET with this radiolabel may provide diagnostic potentials for human neurodegenerative disorders.     

Adrenalectomy Attenuates Kainic Acid-Induced Spectrin Proteolysis and Heat Shock Protein 70 Induction in Hippocampus and Cortex

Abstract: Glucocorticoids have been shown to exacerbate the damaging effects of a variety of neurotoxic insults in the hippocampus and other brain areas. Evidence suggests that the endangering effects of glucocorticoids may be due to augmenting the cascade of events, such as elevations in intracellular calcium levels, because of excitatory amino acid (EAA) receptor stimulation. A potential mechanism responsible for EAA-induced neuronal damage is activation of calcium-sensitive proteases, such as calpain, which then proteolytically degrade cytoskeleton structural proteins, such as spectrin. The present study was designed to determine if glucocorticoids can regulate the spectrin proteolysis produced by the EAA agonist, kainic acid. Rats were adrenalectomized (ADX) or sham operated and 7 days later injected with kainic acid (10 mg/kg). Twenty-four hours later rats were killed and tissues obtained for western blot analyses of the intact spectrin molecule and the proteolytically derived breakdown products. Kainic acid produced an approximate sevenfold increase in the 145–155-kDa spectrin breakdown products in the hippocampus relative to ADX or sham rats injected with vehicle. ADX attenuated the kainic acid-induced increase in breakdown products by 43%. In a similar way, kainic acid produced a large 10-fold increase in spectrin breakdown products in the frontal cortex, which was also significantly attenuated (?80%) by ADX. Induction of heat shock protein 70 (hsp70) by neurotoxic insults has been suggested to be a sensitive indicator of cellular stress in neurons. Kainic acid induced large amounts of hsp70 in both hippocampus and frontal cortex of sham-operated rats that was markedly attenuated (85–95%) by ADX. There was a strong positive correlation between the amount of spectrin proteolysis and the degree of hsp70 induction in both the hippocampus and frontal cortex. In contrast, kainic acid did not significantly produce spectrin proteolysis and induced only a very modest and inconsistent increase of hsp70 in the hypothalamus. This is consistent with the observation that the hypothalamus is relatively insensitive to the neurotoxic effects of systemically administered kainic acid. The dose of kainic acid (10 mg/kg) used in this experiment produces a 10-fold elevation in circulating corticosterone levels at both 1 and 3 h after administration. These results suggest that part of the endangering effects of glucocorticoids on hippocampal and cortical neurons may be due to augmentation of calpain-induced spectrin proteolysis. The attenuation of kainic acid-induced synthesis of hsp70 by ADX indicates that the cellular stress produced by EAAs is regulated in part by glucocorticoids. In addition, the elevation in endogenous corticosterone levels produced by kainic acid appears to be a significant factor contributing to the neuronal damage produced by this agent.     

FORMATION OF GABA FROM PUTRESCINE IN THE BRAIN OF FISH (SALMO IRIDEUS GIBB.)

Abstract— It was demonstrated after intraperitoneal and intracerebral injections of [1,4-¹⁴C]-putrescine.2 HCl that GABA is formed in vivo in the trout brain via a pathway in which glutamic acid is not an intermediate. Intraperitoneal and intracerebral injections of both thiosemicarbazide and 3-mercaptopropionic acid had no measurable effects on GABA concentration, transformation of glutamic acid into GABA in vivo , or on glutamate de-carboxylase activity in the brain within the first 3 h after the application of the inhibitors. Only a small decrease in concentration of pyridoxal phosphate was noticed in the fish brain after thiosemicarbazide administration. The relatively high concentrations of pyridoxal phosphate in the trout brain may be one of the reasons for the ineffectiveness of thiosemicarbazide in inhibiting glutamate decarboxylase in vivo. After intracerebral injections of [1-¹⁴C]GABA, a half-life of 7 h was determined for GABA. The slow turnover rate of GABA in trout brain, which can be assumed from this observation, may give a further explanation of the ineffectiveness of the glutamate decarboxylase inhibitors in lowering the GABA content ot fish brain within a few hours.     

Kainic acid lesions of the median preoptic nucleus: effects on angiotensin II induced drinking and pressor responses in the conscious rat

Input to the nucleus medianus of the preoptic region has been suggested to be involved in both the drinking and pressor responses elicited by the central administration of angiotensin II. Evidence in support of this suggestion has been gained principally from electrical lesion experiments. This lesion procedure does not differentiate between the cells of the region and fibers coursing through the region. To test the hypothesis that cells in this region are involved in both the pressor and drinking responses elicited by central administration of angiotensin II, injections of kainic acid were made to induce lesions of the cells, while sparing fibers of passage. Drinking and blood pressure responses were determined pre- and post-lesion in the chronically instrumented awake rat. Injections of 50 ng angiotensin II in a 2-microL volume into a lateral cerebral ventricle of the conscious rat elicited pronounced drinking and pressor responses with a latency of 3-5 min. Lesions of the median preoptic region produced by injecting 1.0 microgram of kainic acid in 0.25 microL for 15 s attenuated or blocked the drinking response and increased the latency to drink induced by central injections of angiotensin II. However, kainic acid lesions did not significantly alter the pressor responses produced by angiotensin II administration. These results suggest that cells in the median preoptic region are involved in the drinking response but do not participate in the pressor response elicited by angiotensin II administration into a lateral cerebral ventricle of the conscious rat.     

Retrograde degeneration of basal forebrain cholinergic neurons after neurotoxin lesions of the neocortex: application of ganglioside GM1

Injections of the neurotoxin kainic acid were made unilaterally at multiple loci in the cerebral cortex of the rat in an attempt to reproduce aspects of the central pathology of Alzheimer's disease. Neurochemical markers of cholinergic and GABAergic function in the cortex and basal forebrain, determined after various intervals, suggested that subsequent to initial destruction of cortical neuronal cell bodies, trans-synaptic retrograde degeneration of cholinergic neurons occurred in the nucleus basalis magnocellularis (NBM) projecting to the cortex.

Contrary to the situation noted after devascularizing cortical lesions, there was no spontaneous recovery from this effect of kainic acid in the ipsilateral NBM. Similarly, these retrograde effects could not be prevented by the administration of the ganglioside GM₁. These observations suggest that kainic acid compromises the plastic capacity of this cholinergic projection, perhaps by affecting the production of endogenous trophic factors. This may be of relevance in developing the use of neurotoxins for models of neurodegenerative disease.     

Decreased Expression of Calmodulin Kinase II and Calcineurin Messenger RNAs in the Mouse Hippocampus After Kainic Acid-Induced Seizures

Abstract: The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are especially abundant in the mammalian CNS, where they have been implicated repeatedly in different neuronal functions. CaMKII is a holoenzyme that is likely to be constituted of both homomultimers and heteromultimers, CaMKIIα and CaMKIIβ being the most abundant subunits in the brain. CaN is a heterodimer constituted of a catalytic subunit (CaN A) and a regulatory subunit (CaN B), and CaN Aα is the predominant form in the brain. We studied the expression of CaMKIIα, CaMKIIβ, and CaN Aα subunit messenger RNAs in the mouse hippocampus at different times after the administration of a convulsant dose of kainic acid. CaMKIIα and CaN A immunohistochemistry was also performed. We observed a transient decrease in the three messenger RNAs in the kainic acid-treated mice, peaking at 5 or 24 h of treatment. The effect had disappeared completely 8 days after treatment. No significant alterations in CaMKII or CaN immunolabelling were observed in the hippocampus of kainic acid-treated mice. The observed modifications could be due to the neuronal hyperexcitability induced by kainic acid rather than neuronal degeneration, because no areas of neuronal loss were detected. Our results suggest that the expression of CaMKII and CaN mRNAs is down-regulated in neuronal cells in response to the hyperexcitability induced by kainic acid. The transient nature of the effect and the apparent absence of significant modifications in the amount of their corresponding proteins may be related to the absence of neuronal damage.     

Obesity induced by kainic acid microlesion in the ventromedial hypothalamus in rats

The neurotoxin kainic acid was injected bilaterally into the ventromedial hypothalamus of female rats in doses of 100 or 200 ng. The injections produced microlesions which led to progressive body weight gain and fat deposition in dose dependent manner. The histological examination revealed that the lesions were mainly located in the region between the ventromedial hypothalamic nuclei and the fornix.     

Comparative study of the epileptogenic effect of kainic acid injected into the cerebral cortex, hippocampus and amygdala in adult cats chronically implanted

Intracerebral injection of kainic acid in cerebral cortex, hippocampus or amygdala in cats chronically implanted showed that: 1) Hippocampus and amygdala presented a greater sensitivity than the cerebral cortex, while hippocampus presented a greater sensitivity than the amygdala to the generation of an epileptic focus. 2) Comparison of latency, mean duration of afterdischarges, and the mean time period to obtain the peak intensity of the afterdischarge in the three cited structures, showed that mean latency of the first afterdischarge was significantly shorter in hippocampus and amygdala compared with the cerebral cortex. Moreover the mean time period to reach the peak intensity of the afterdischarge was again shorter in the subcortical structures. 3) The epileptic foci both in hippocampus and amygdala were blocked by CNQX and muscimol. 4) The behavioral changes depended on the intensity of the epileptic process. Tonic-clonic convulsions appeared only when the motor cerebral cortex was involved. Finally, 5) kainic acid injections in hippocampus and amygdala elicited an intense neuronal destruction and gliosis of these structures. We conclude that intracerebral injection of low doses of kainic acid in cats represent a good model to study focal epileptic thresholds in the CNS.     

Involvement of Free Radicals in Excitotoxicity In Vivo

Abstract: Recent evidence has linked excitotoxicity with the generation of free radicals. We examined whether free radical spin traps can attenuate excitotoxic lesions in vivo. Pretreatment with N-tert -butyl-α-(2-sulfophenyl)-nitrone (S-PBN) significantly attenuated striatal excitotoxic lesions in rats produced by N -methyl- d -aspartate (NMDA), kainic acid, and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). In a similar manner, striatal lesions produced by 1-methyl-4-phenylpyridinium (MPP⁺), malonate, and 3-acetylpyridine were significantly attenuated by either S-PBN or α-phenyl- N-tert -butylnitrone (PBN) treatment. Administration of S-PBN in combination with the NMDA antagonist MK-801 produced additive effects against malonate and 3-acetylpyridine toxicity. Malonate injections resulted in increased production of hydroxyl free radicals (^•OH) as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (DHBA). This increase was significantly attenuated by S-PBN, consistent with a free radical scavenging effect. S-PBN had no effects on malonate-induced ATP depletions and had no significant effect on spontaneous striatal electrophysiologic activity. These results provide the first direct in vivo evidence for the involvement of free radicals in excitotoxicity and suggest that antioxidants may be useful in treating neurologic illnesses in which excitotoxic mechanisms have been implicated.     

Correlation between rotational behaviors and neurochemical changes associated with damage of rat striatum: Analysis using unilateral microinjection of kainic acid

The correlation between rotational behaviors and neurochemical changes associated with the striatal damage induced by an unilateral microinjection of kainic acid were investigated. Shortly after the unilateral striatal injection of kainic acid, rats exhibited contralateral rotational behaviors, and these changes were antagonized by the simultaneous striatal injection of haloperidol. On the other hand, systemic injection of methamphetamine to animals having the lesion on nigro-striatal dopaminergic neurons exhibited ipsilateral turnings. In addition, it was found that the release of [¹⁴C]dopamine from striatal slices was increased by the in vitro addition of kainic acid. Following 2 days after the striatal injection of kainic acid and thereafter, the rats exhibited ipsilateral rotational behaviors and microinjection of muscimol into the ipsilateral substantia nigra of these animals altered turning movements to a contralateral type. Simultaneous nigral injection of bicuculline antagonized to the muscimol-induced contralateral turnings. These results suggest that the increase of dopamine release from dopaminergic neurons in the striatum may be involved in the occurrence of contralateral turning behaviors observed shortly after the striatal kainic acid treatment. The present results also suggest that changes in the functional states of striatonigral GABA-ergic neurons may play an important role in the occurrence of ipsilateral rotational movements at a late stage following the striatal injection of this agent.     

Pronounced increases in brain levels of calcitonin gene-related peptide after kainic acid induced seizures

Changes in immunoreactivity of calcitonin gene-related peptide (CGRP) were investigated in the brains of rats subsequently to seizures induced by intraperitoneal injection of kainic acid (10 mg/kg, i.p.). Increased levels of the neuropeptide were observed in the frontal cortex (increase of 1300% of control value), striatum (900%), dorsal hippocampus (400%) and amygdala/pyriform cortex (135%) three days after injection of the neurotoxin. Intravenous infusion of mannitol (1.5 g/kg, under thiopental anesthesia) which prevents seizures and post-seizure brain damage suppressed the changes in CGRP-like immunoreactivity. Injection of pentylenetetrazol causing generalized motor seizures resulted in no change of CGRP-immunoreactivity after three days. The pronounced but reversible increases of brain CGRP levels suggest a strong but short-lasting activation of the peptide system. The failure of pentylenetetrazol to produce a similar effect and the protective action of mannitol suggest that sustained seizures and/or post-seizure brain damage may be required to produce the rise in peptide levels.     

Does phenobarbital protect against trimethyltin-induced neuropathology of limbic structures?

Because of the similarity in the pattern of limbic sites damaged by both compounds, it has been suggested that trimethyltin (TMT) may be an excitotoxin like kainic acid (KA). KA produces seizures which eventually result in neuronal damage similar to that found in epilepsy. Anticonvulsants reduce both the seizures and pathology associated with KA. Because TMT may also produce seizures, we undertook to determine whether or not some of the TMT-induced limbic neuropathology could result from seizure activity. To do this, a single dose of TMT chloride (either 7.5 or 15 mg/kg) was given per os to rats, and then phenobarbital (30 mg/kg) was administered subcutaneously in repeated doses. Treatment with phenobarbital did not prevent pathologic changes in the hippocampus, dentate gyrus, and pyriform or prepyriform cortex. Since phenobarbital did not protect against TMT-induced neuronal damage, as it has been reported by others to protect against KA-induced damage, the present findings suggest that these two toxicants probably produce hippocampal pathology via different mechanisms and that the TMT-induced pathologic changes do not require sustained electrical seizure activity.     

Identification of medullary loci critical for neurogenesis of gasping

We hypothesized that a discrete medullary locus, critical for gasping neurogenesis, could be identified. In decerebrate, cerebellectomized, vagotomized, paralyzed, and ventilated cats, activities of phrenic, hypoglossal, and recurrent laryngeal nerves were monitored. Gasping was induced by freezing the brain stem, via a fork thermode, at the pontomedullary junction. By reversible cooling of the medulla, chemical lesions with kainic acid, and radio-frequency lesions, a critical area for gasping neurogenesis was localized bilaterally 2-3 mm rostral to obex, 2.0-2.5 mm lateral to midline, and 3-4 mm ventral to medullary surface. Electrical stimulation in this area elicited premature gasps, whereas unilateral lesions or lidocaine injections eliminated gasping activities in all nerves. These procedures did not cause similar changes during eupnea. In apneusis, however, lidocaine injections markedly altered the pattern or caused apnea. We conclude that discharge of neurons in a discrete portion of the lateral tegmental field of medulla is required for gasping neurogenesis. Our results are consistent with these neurons comprising the central pattern generator for gasping.