Amygdaloid Stimulation Research Articles

A sub-threshold dose of pilocarpine increases glutamine synthetase in reactive astrocytes and enhances the progression of amygdaloid-kindling epilepsy in rats.

The prognosis of patients exposed to a sub-threshold dose of a proconvulsant is difficult to establish. In this study, we investigated the effect of a single sub-threshold dose of the proconvulsant pilocarpine (PILO) on the progression of seizures that were subsequently induced by daily electrical stimulation (kindling) of the amygdaloid formation. Male Sprague–Dawley rats were each implanted with an electrode in the right basolateral amygdala and an indwelling cannula in the right ventricle. The animals were randomized into groups and were administered one of the following treatments: saline, PILO, saline+L-α-aminoadipic acid (L-AAA; one dosage tested), PILO+L-AAA, or PILO+L-methionine sulfoximine (three dosages tested). Amygdaloid stimulation and electroencephalography were performed once daily. We performed immunohistochemistry and western blot for glial fibrillary acidic protein and glutamine synthetase (GS). We also assayed the enzymic activity of GS in discrete brain regions. An intraperitoneal injection of a sub-threshold PILO dose enhanced the progression of amygdaloid-kindling seizures and was accompanied by an increase in reactive-astrocyte and GS (content and activity) in the hippocampus and piriform cortex. L-AAA and L-methionine sulfoximine, inhibitors of astrocytic and GS function, respectively, abolished the effect of PILO on amygdaloid-kindling seizures. We conclude that one sub-threshold dose of a proconvulsant may enhance the progression of subsequent epilepsy and astrocytic GS may play a role in this phenomenon. Thus, a future therapy for epilepsy could be inhibition of astrocytes and/or GS.

Localized cortical injections of ethosuximide suppress spike-and-wave activity and reduce the resistance to kindling in genetic absence epilepsy rats (GAERS)

Models of genetic absence epilepsy are resistant to secondary generalization of focal limbic seizures. This correlates with the postnatal development of spike-and-wave discharges (SWDs), a hallmark of absence seizures arising from a cortical focus in the perioral region of somatosensory cortex. Ethosuximide injected at this site suppresses SWDs. The effect of this suppression on kindling in "Genetic Absence Epilepsy Rats from Strasbourg" (GAERS), has been compared for postnatal 30 day (PN30) rats having immature SWDs and adult (>4 months) rats having mature SWDs. Non-epileptic Wistar and GAERS rats were implanted with a basolateral amygdaloid stimulation electrode, bilateral injection cannulas into the cortical perioral focus, and cortical recording electrodes. Following recovery cortical injections of ethosuximide or saline were made and after 30min rats were given 36 stimulations or until Racine's stage 5 seizures were produced. All Wistar rats (PN30 and adult) treated with saline or ethosuximide reached stage 5. Of GAERS given saline, 33% (PN30) and 43% (adults) were resistant to kindling; after ethosuximide pups behaved like Wistars, but adults showed a delay in kindling relative to Wistars. These findings imply that mechanisms underlying kindling resistance are related but not limited to SWD activity in animals with genetic absence epilepsy.

Mossy fiber sprouting is dissociated from kindling of generalized seizures in the guinea-pig.

Controversy surrounds whether aberrant mossy fiber sprouting in the hippocampus is necessary for the establishment of seizure states. We investigated the association between mossy fiber sprouting and kindling in guinea-pigs, using either single-site or alternate-site stimulation. Kindling with single-site amygdaloid stimulation did not induce significant sprouting, despite the development of partial seizures. In contrast, single-site septal and alternating amygdaloid-septal stimulation produced moderate but significant sprouting in about 60% of animals that failed to develop stage 5 generalized seizures. Since the magnitude of sprouting was similar despite striking differences in the intensity of seizures that developed, we conclude that mossy sprouting is not causally associated with seizure development.

Time course of c-FOS expression in status epilepticus induced by amygdaloid stimulation.

Decaying of c-FOS immunoreactivity (FIR) was studied in adult rats with 1 h self-sustaining limbic status epilepticus (SSLSE) induced by amygdaloid electrical stimulation. Rats that failed to enter SSLSE showed localized FIR in the ipsilateral limbic cortex, neocortex, and amygdala. FIR became bilaterally extensive, including the hippocampal formation 0.5 h after SSLSE. It decreased gradually between 2 and 6 h and returned to basal levels around 1 day. Neocortical FIR in clonic SSLSE persisted longer than in other types of SSLSE. We demonstrate for the first time that FIR in SSLSE lasts much longer than several hours, its decaying is related to the seizure behavior, and absent or weak FIR at the hippocampal formation is associated with failed SSLSE entry.

Electrical self-stimulation in the central amygdaloid nucleus after ibotenic acid lesion of the lateral hypothalamus

This experiment was carried out in order to investigate the involvement of lateral hypothalamus (LH) in electrical self-stimulation of the central amygdaloid nucleus (CeA). Adult male Sprague-Dawley rats were bilaterally implanted with a guide cannula situated above each LH and with two electrodes in the CeA. Self-stimulation was subsequently obtained separately from both right and left electrodes. The LH was then lesioned unilaterally by ibotenic acid (IBO) injection. Eight days later, the effect of this unilateral lesion on self-stimulation of the ipsilateral and contralateral CeA was tested. Then the neurons of the remaining non-lesioned LH side were lesioned with IBO and self-stimulation was tested 15 days after the second lesion. Both unilateral as well as bilateral lesions of LH produced a significant decrease in CeA self-stimulation rates but had no significant effect on the reward effectiveness. The unilateral lesions did not produce any modification of the rate-intensity function in the contralateral CeA. This lesion-induced depression in performance was reversed by treatment with phenobarbital. These results provide clear evidence that the rewarding effects of CeA electrical stimulation do not result from the activation of the LH outputs and that the apparent decrease in CeA self-stimulation may result from the LH lesion-induced increase in the frequency of epileptiform manifestations that occur following amygdaloid stimulation.

Ethanol enhances medial amygdaloid induced inhibition of predatory attack behaviour in the cat: role of GABAA receptors in the lateral hypothalamus.

The present study tested the hypothesis that the suppressive effects of ethanol upon predatory attack behaviour in the cat involve a pathway from the medial amygdala to the lateral hypothalamus, and that these suppressive effects are mediated by gamma-aminobutyric acid (GABAA) receptors located in the lateral hypothalamus. Cannula electrodes were implanted into the lateral hypothalamus for elicitation of predatory attack behaviour and for microinjections of the GABAA receptor antagonist, bicuculline. Monopolar stimulating electrodes were implanted into the medial amygdala from which subseizure levels of electrical stimulation suppressed predatory attack behaviour. In the first phase of the study, we compared response latencies for predatory attack behaviour following single stimulation of the lateral hypothalamus alone with those following paired trials of dual stimulation of the medial amygdala plus lateral hypothalamus. Dual stimulation significantly suppressed predatory attack. In the second phase of the study, peripheral ethanol administration (in doses of 0.01, 0.5 and 1.0 g/kg, i.p.) enhanced the suppressive effects of medial amygdaloid stimulation in a dose- and time-dependent manner in which peak effects were obtained 60 min post-injection. In the third phase of the study, bicuculline (0.15 nmol) was microinjected into the lateral hypothalamus both prior to and following paired trials of dual stimulation. Drug infusion blocked the suppressive effects of medial amygdaloid stimulation upon predatory attack behaviour elicited from the lateral hypothalamus, indicating the importance of GABAA receptors in mediating this suppression. In the fourth phase of the study, bicuculline, microinjected into the lateral hypothalamus at the time when ethanol's effects were maximal (i.e. 60-80 min post-ethanol administration), totally blocked the suppressive effects of medial amygdaloid stimulation as well as the enhancing effects of ethanol upon medial amygdaloid suppression of this form of aggressive behaviour. In the last phase of the study, bicuculline (0.15 nmol) infusion into the lateral hypothalamus significantly reduced the suppressive effects of ethanol (1.0 g/kg, i.p.) upon predatory attack behaviour elicited from the lateral hypothalamus. These results support the hypothesis that ethanol's suppressive effects upon predatory attack behaviour in the cat are mediated, at least in part, by GABAA receptors in the lateral hypothalamus. The present and recent findings in our laboratory support the view that GABAA receptors in the lateral hypothalamus are activated, in turn, by a GABAergic pathway which arises from the medial hypothalamus whose neurons receive inputs from the medial amygdala.

Medial amygdaloid suppression of predatory attack behavior in the cat: II. Role of a GABAergic pathway from the medial to the lateral hypothalamus

The medial amygdala is known to powerfully suppress predatory attack behavior elicited by electrical stimulation of the lateral hypothalamus of the cat. In the preceding paper, it was shown that the initial limb of a pathway subserving suppression of predatory attack from the medial amygdala to the lateral hypothalamus projects to the ventromedial hypothalamus and its functions are mediated by substance P. The present study tested the hypothesis that the second limb of the pathway subserving medial amygdaloid suppression of predatory attack behavior projects from the medial to lateral hypothalamus and its functions are mediated by GABA. Cannula electrodes were implanted into the lateral hypothalamus for elicitation of predatory attack behavior as well as for the microinfusion of GABA compounds. Monopolar stimulating electrodes were implanted into sites within the medial amygdala from which subseizure levels of stimulation could suppress predatory attack behavior. Initially, the effects of dual stimulation of the medial amygdala and lateral hypothalamus upon response latencies for predatory attack were compared with single stimulation of the lateral hypothalamus alone. Dual stimulation was shown to significantly suppress predatory attack elicited from the lateral hypothalamus. Then, the GABA A receptor antagonist, bicuculline, was microinjected into sites within the lateral hypothalamus from which predatory attack was elicited in doses of 0.015, 0.075 and 0.15 nmol and paired trials of single and dual stimulation were again repeated in a manner identical to that applied prior to drug administration. Drug infusion produced a blockade of medial amygdaloid suppression of predatory attack in a time- and dose-dependent manner. Conversely, microinfusions of the GABA A receptor agonist, muscimol (10, 25 and 50 pmol), into the same lateral hypothalamic ‘attack’ site in the absence of medial amygdaloid stimulation suppressed predatory attack, thus simulating the effects of medial amygdaloid stimulation. Furthermore, pretreatment with bicuculline microinjected into the lateral hypothalamus blocked the suppressive effects of substance P, that was infused into the ventromedial hypothalamus, upon predatory attack. Receptor autoradiography demonstrated the presence of high affinity binding for GABA A receptors in the lateral hypothalamus. A combination of immunocytochemical and retrograde axonal tract tracing procedures, in which Fluoro-Gold was microinjected into the lateral hypothalamic attack sites, revealed the presence of populations of neurons labeled for both Fluoro-Gold and GABA in the ventromedial hypothalamus. These findings provide new evidence for the existence of a pathway from the medial to lateral hypothalamus whose functions are mediated by GABA. Thus, the overall findings provide support for the view that the pathway from the medial amygdala to the lateral hypothalamus underlying suppression of predatory attack behavior involves a two-neuronal arc: the first neuron projects from the medial amygdala to the medial hypothalamus and its functions are mediated by substance P; the second neuron involves a GABAergic pathway originating in the ventromedial hypothalamus and which projects to the lateral hypothalamus.

Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus

A novel murine cDNA which encodes a protein designated neuropsin was cloned. Northern and in situ hybridization analyses demonstrated that neuropsin mRNA is expressed specifically in the limbic system of mouse brain and is localized at highest concentration in pyramidal neurons of the hippocampal CA1-3 subfields. Direct hippocampal stimulation and kindling induced by amygdaloid stimulation caused a significant bilateral change in neuropsin mRNA level in the hippocampal pyramidal neurons. The activity-dependent changes and the specific localization indicate that neuropsin is involved in hippocampal plasticity.

Neurochemical mechanisms underlying amygdaloid modulation of aggressive behavior in the cat

Studies designed to determine the respective roles of substance P, excitatory amino acids, and enkephalins in amygdaloid modulation of defensive rage behavior in the cat are presented. The basic design of these studies involved three stages. In stage I, cannula electrodes for stimulation and drug infusion were implanted into medial hypothalamic of midbrain periaqueductal gray (PAG) sites from which defensive rage behavior could be elicited. Then, a stimulating electrode was implanted into a site within the medial, basal, or central nuclear complex from which modulation of the defensive rage response could be obtained. Amygdaloid modulation of defensive rage was determined in the following manner: it employed the paradigm of dual stimulation in which comparisons were made of response latencies between alternate trials of dual (i.e., amygdala+medial hypothalamus [or PAG]) and single stimulation of the hypothalamus or PAG alone. Thus, stage I established the baseline level of modulation (i.e., facilitation or suppression of defensive rage) in the predrug stimulation period. In stage II, a selective or non-selective receptor antagonist for a given transmitter system was administered either peripherally or intracerebrally at the defensive rage site, after which time the same dual stimulation paradigm was then repeated over the ensuing 180 min postinjection period in order to determine the effects of drug delivery upon amydaloid modulation of defensive rage. Stage III of the study took place at the completion of the pharmacological testing phase. The retrograde axonal tracer, Fluoro-Gold, was microinjected into the defensive rage site within the medial hypothalamus or PAG, and following a 6-14 day survival period, animals were sacrificed and brains were processed for histological and immunocytochemical analyses for the neurotransmitters noted above. This procedure thus permitted identification of cells within the amygdala which were labeled retrogradely and which were also immunostained positively for substance P, excitatory amino acids, or enkephalin. For studies involving substance P, defensive rage was elicited from the medial hypothalamus and for studies examining the roles of excitatory amino acids and enkaphalin, defensive rage was elicited from the PAG. In the first study, facilitation of hypothalamically elicited defensive rage was obtained with dual stimulation of the medial nucleus of the amygdala. In separate experiments, the selective NK 1 non-peptide antagonist, CP 96,345, was administered both peripherally as well as intracerabrally into the hypothalamic defensive rage sites in doses of 0.5-4.0 mg/kg (i.p.) and 0.5-2.5 nmol (i.c.). Following drug delivery, the facilitatory effects of medial amygdaloid stimulation were blocked ina dose- and time-dependent manner in which the effects were noted as early as 5 min postinjection. The maximum drug dose (4.0 mg/kg) employed for peripheral administration resulted in a 42% reduction in the facilitatory effects of the medial amygdala (P<0.002). This drug, when microinjected directly into medial hypothalamic defensive rage sites at the maximum dose level of 2.5 nmol, resulted in an 84% reduction of the suppressive effects of amygdaloid stimulation (P<0.5) at 5 min postinjection. In the next study, an N-methyl-D-aspartate (NMDA) antagonist, DL-α-amino-7-phosphonoheptanoic acid (AP-7), was administered either peripherally (0.1-1.0 mg/kg) or intracerebrally (0.2 and 2.0 nmol) into PAG defensive rage sites

Relationship between atrophy of the amygdala and ictal fear in temporal lobe epilepsy.

Viscerosensory and affective manifestations are often elicited by temporal lobe seizure discharges. They have been reproduced by amygdaloid stimulation in awake patients during stereotaxic exploration or neurosurgical procedures. They are not exclusively reproduced by stimulation of the amygdala, though most commonly they are evoked from it. Ictal fear is frequently, but not invariably, associated with a rising epigastric sensation, palpitations, mydriasis and pallor. We studied 50 patients (mean age 33 years) with intractable temporal lobe epilepsy (TLE): MRI volumetric measurements of amygdala and hippocampus were performed using a protocol previously described by our group (Watson et al., Neurology 1992; 42: 1743-50). All patients had extensive EEG investigation and at least two seizures recorded by video-EEG monitoring. Seventeen patients (34%) had a clear history of fear accompanied by a rising epigastric sensation as the initial manifestation of their habitual attacks. The amygdala volumes in this group were significantly (P = 0.001) smaller (mean 2131.6 mm3) compared with the volumes of the 33 patients without these symptoms (mean 2561.5 mm3). Both patient groups had smaller mean amygdala volumes compared with normal controls (mean 2828.2 mm3). Postoperative pathology correlated well with volumetric atrophy. In addition, we found that patients with more pronounced amygdaloid atrophy more commonly had prolonged febrile convulsions in early childhood and also more frequently secondarily generalized seizures. Results support the finding that ictal fear is related to pathology of the amygdala and that it, like the hippocampus, is an important substrate of TLE.

Basal amygdaloid facilitation of midbrain periaqueductal gray elicited defensive rage behavior in the cat is mediated through NMDA receptors

The present study tested the hypotheses that: (1) defensive rage behavior eelicited from the midbrain periaqueductal gray (PAG) in the cat is facilitated from the basal complex of amygdala; and (2) such facilitation from this region of amygdala is mediated via a pathway in which excitatory amino acids acting upon NMDA receptors within the PAG are utilized as a neurotransmitter. In the first phase of this study, cannula electrodes were implanted into PAG sites for the elicitation of defensive rage behavior as well as for drug delivery. Then, a second monopolar electrode was implanted into the basal nucleus of amygdala from which facilitation of defensive rage could be obtained. As a esult of dual stimulation of the basal amygdala and PAG, response latencies for defensive rage were significantly lowered relative to PAG stimulation alone (P < 0.01). In the second phase of this experiment, 3 doses of a selective NMDA receptor antagonist, AP-7 (0.1, 0.5, 1.0 mg/kg), were peripherally (i.p.) administered in 5 animals. The results indicated a significant decrease in the facilitatory effects of amygdaloid stimulation in a dose and time dependent manner (P < 0.001). In the third phase, AP-7 was administered intracerebrally into PAG defensive rage sites in dose of 0.2 and 2.0 nmol. It was noted that intracerebral microinjections of AP-7 at the higher dose (2.0 nmol) also significantly suppressed the facilitatory effects of amygdaloid stimulation (P < 0.01); however, these effects were somewhat less potent then those observed following peripheral drug administration. A fourth phase of the study was conducted at the completion of the pharmacological experiments. Here, retrograde axonal tracing of amygdaloid neurons projecting to the PAG following Fluoro-Gold microinjections placed in defensive rage sites was coupled with an immunocytochemical analysis of the distribution of excitatory amino acid positive neurons present within the basal amygdala. The findings revealed the presence of high concentrations of glutamate and asparate positive cells located mainly within the basal and lateral nuclei of amygdala, while retrogradely labeled cells within the amygdala were confined to its basal complex, central nucleus and anterior amygdaloid area. More significantly, dense quantities of cells double labeled with both Fluoro-Gold and excitatory amino acids were identified within the basal complex of amygdala. Collectively, these findings support the view that neurons arising from the basal complex of amygdala serve to facilitate defensive rage behavior, in part, by virtue of their projections to the PAG and that this phenomenon is mediated by a mechanism utilizing excitatory amino acids that act through NMDA receptors.

Effect of medial amygdaloid stimulation on pancreatic exocrine secretion in anesthetized rats.

Effects of medial amygdaloid stimulation on pancreatic exocrine secretion were investigated in anesthetized rats with pyloric ligation. During intraduodenal 0.01 N HCl perfusion, the electrical stimulation of the medial amygdaloid nucleus augmented significantly the HCl-induced pancreatic secretion of water and bicarbonate output. The duodenal acidification significantly increased secretin concentration in plasma. The effects of medial amygdaloid stimulation were also tested in the rats that received a small dose of porcine secretin. Although the amount of secretin infused was not enough to increase the pancreatic exocrine secretion, the stimulation of the medial amygdaloid nucleus significantly increased flow and bicarbonate output. The augmented pancreatic secretory responses to electrical stimulation of the medial amygdaloid nucleus were abolished by bilateral truncal vagotomy. However, the medial amygdaloid stimulation was not effective in stimulating pancreatic secretion in the rats perfused with isotonic saline. From these results, it is suggested that the medial amygdaloid nucleus plays a stimulatory role in pancreatic exocrine secretion via vagus nerves and that a certain level of plasma secretin is needed for the medial amygdaloid stimulation to be effective.

Evidence that substance P is utilized in medial amygdaloid facilitation of defensive rage behavior in the cat

The present study was designed to test the hypothesis that a major excitatory mechanism for the expression of feline defensive rage behavior involves the medial nucleus of the amygdala which utilizes substance P as a neurotransmitter in a direct output pathway that supplies the medial hypothalamus. In phase I of the experiment, stimulating electrodes were implanted into the medial amygdala and cannula electrodes were implanted into the medial and lateral hypothalamus from which defensive rage and predatory attack behavior could be elecited by electrical stimulation, respectively. Response latencies for defensive rage were significantly lowered after dual stimulation of the medial amygdala and medial hypothalamus relative to single stimulation of the medial hypothalamus alone. In phase II, dose- and time-dependent decreases in medial amygdaloid-induced facilitation of defensive rage were observed after the i.p. administration of the NK 1 antagonist, CP-96,345 (0.05, 2 and 4 mg/kg). In phase III of the study, the effects of microinjections of CP-96,345 placed directly into defensive rage sites within the medial hypothalamus (0.05, 0.5 and 2.5 nmol) upon medial amygdaloid modulation of this response were assessed. Again, intracerebral administration of this antagonist blocked the facilitatory effects of medial amygdaloid-induced facilitation of defensive rage in a manner parallel to that observed with peripheral administration of the NK 1 antagonist. The results suggest that the medial amygdala facilitates defensive rage by acting through a substance P mechanism at the level of the medial hypothalamus. Other experiments revealed that peripheral administration of the NK 1 antagonist: (1) had little effect upon the latency or threshold for elicitation of defensive rage, suggesting that the medial amygdaloid-substance P facilitatory mechanism acts in a phasic rather than tonic manner; and (2) also blocks the suppressive effects of medial amygdaloid stimulation upon predatory attack behavior elicited from the lateral hypothalamus. The latter finding suggests that similar neurochemical mechanisms regulate medial amygdaloid modulation of both forms of hypothalamically elicited aggression. The final aspect of this study utilized the combination of retrograde-tracing of amygdaloid neurons into the medial hypothalamus after microinjections of Fluoro-Gold into defensive rage sites, and the immunocytochemical analysis of substance P neurons within the amygdala. The data indicated that large numbers of retrogradely and immunocytochemically positive labeled cells were identified in the medial nucleus, including many that were double-labeled. The overall findings of this study support the hypothesis that the medial amygdala provides a powerful excitatory monosynaptic input to the medial hypothalamus for the expression of defensive rage behavior and that this mechanism is mediated, at least in part, via a substance P mechanism.

Effects of scopolamine on the rewarding and seizure-inducing properties of amygdaloid stimulation

The relationship between amygdaloid brain-stimulation reward and the evolution of seizure activity was evaluated in this study. Current levels that maintained optimal intracranial self-stimulation (ICSS) rates were found to be lower than the minimal current intensity required to elicit an afterdischarge (AD) from the central nucleus of the amygdala. After the ICSS session, AD thresholds (ADTs) were reduced to the same levels of current used to support ICSS. Assessment of seizure stage development during ICSS testing revealed that the emergence of early-stage epileptiform events following repeated amygdaloid stimulation suppressed ICSS performance. While administration of the antimuscarinic scopolamine did not prevent the stimulation-elicited reduction in AD thresholds, it was observed to inhibit seizure progression and increase ICSS rates. These results are consistent with the excitatory function of acetylcholine in epileptogenesis and were related to the possibility that different mechanisms underlie the rewarding and seizure-inducing properties of amygdaloid stimulation.

The effects of amygdaloid stimulation on amphetamine-elicited locomotor sensitization

Systemic injection of d-amphetamine (1.0 mg/kg) resulted in a progressive increase in locomotor activity as a function of repeated daily drug administration. The magnitude of the stimulant-induced sensitization effect was enhanced by low-current electrical stimulation of the central nucleus of the amygdala during open-field testing. Amygdaloid stimulation in the absence of amphetamine treatment did not influence spontaneous locomotor activity, and there was no behavioral evidence of epileptogenesis following amygdaloid stimulation over the course of the experiment. However, with continued stimulation of the amygdala, early-stage convulsive activity was apparent in animals after approximately 40 days of testing, signifying the advancement of kindling evolution. These results suggest that the processes responsible for kindling acquisition, prior to the behavioral expression of epileptiform events, interact with the underlying substrates of amphetamine sensitization.

Effects of systemic glucose injection on the development of amygdala kindling in rats

The effects of systemic glucose administration on the development of electric amygdaloid kindling seizures were examined in Wistar rats. Daily intraperitoneal injections of glucose 100 mg/kg, 20 min prior to each amygdaloid stimulation did affect the rate of kindling development in comparison with the saline-injected group. The number of stimulations required to reach stage 5 was lower in animals treated with glucose. The facilitation of kindling development found in glucose-treated rats is related to a specific decrease in the number of stimulations needed to evolve from stage 2 to stage 3. These results indicate that systemic glucose administration facilitates amygdaloid kindling in rats.

Alterations in somatostatin and proenkephalin mRNA in response to a single amygdaloid stimulation versus kindling

Previous studies have shown changes in both somatostatin (SS)- and proenkephalin(PE)-derived peptides in the brains of amygdaloid-kindled rats, suggesting possible roles for the peptides in the kindling process. In this study, we have extended this analysis by looking at the time course of changes in SS and PE mRNAs at various times after kindling, in comparison with a single non-convulsive stimulation. Blot analysis of total RNA showed increases in SS mRNA in striatum, frontal cortex and hippocampus of animals receiving only a single stimulation as well as kindled animals — the increase occurred 1–3 days following stimulation and levels were back to basal by 1 week. PE mRNA did not change. In situ hybridization analysis, one day after the last kindling stimulation, showed significant elevations of SS mRNA in CA1, CA2 and dentate gyrus of hippocampus and of PE mRNA in olfactory cortex that were specific to kindling. However, both a single stimulation and kindling increased PE mRNA in olfactory tubercle and arcuate nucleus. In contrast, a single electrical stimulus increased PE mRNA in ventral striatum and SS mRNA in cingulate cortex and olfactory tubercle. These data support the idea that changes of SS mRNA in hippocampus and of PE mRNA in olfactory cortex may be related to kindling, and point out the importance of using animals which receive a single electrical stimulus, rather than sham-operated animals, as controls.

An enkephalinergic mechanism involved in amygdaloid suppression of affective defence behavior elicited from the midbrain periaqueductal gray in the cat

A series of recent studies in our laboratory have provided evidence that opioid peptides powerfully suppress feline affective defense behavior at the level of the midbrain periaqueductal gray (PAG). In the present study, we tested the hypothesis that the central (CE) nucleus of the amygdala constitutes a significant inhibitory input to the PAG which utilizes enkephalins as its neurotransmitter or neuromodulator. Cannula-electrodes were implanted into the PAG for the elicitation of affective defense behavior as well as for infusion of opioid antagonists. Monopolar stimulating electrodes were also implanted into the central, lateral and medial amygdaloid nuclei from which suppression or facilitation of affective defense behavior could be obtained. Initially, 4 trials of concurrent, subseizure stimulation of the CE or lateral amygdala at very low (100 μA, 60 Hz) currents and PAG resulted in an immediate suppression of this response which displayed a time dependent decline after 30 min. In the next stage of the experiment, naloxone (2.7, 18.9 and 27.5 nM) was microinjected through the cannula-electrode into the PAG affective defense site and the experimental procedures noted above were repeated. Naloxone treatment (at 27.5 and 18.9 nM) blocked the suppressive effects of CE and lateral amygdaloid stimulation in a dose and time dependent manner. Further analysis revealed that this effect is likely mediated via the μ receptor since the suppressive effects of amygdaloid stimulation were blocked by the selective μ antagonist, β-Funaltrexamine (0.05 and 0.2 nM) but not by the selective σ-antagonist, ICI 174,864 (0.7 nM). Moreover, facilitation of affective defense behavior following dual stimulation of the medial amygdala and PAG was not affected by naloxone administration (27.5 nM). The final stage of the study was designed to further test the hypothesis that neurons in the CE contain cell bodies that are both enkephalinergic and project to the dorsal aspect of the PAG associated with the expression of affective defense behavior. This aspect involved the combination of retrograde tracing of amygdaloid neurons into the PAG following microinjections of the retrograde tracer, Fluoro-Gold, and the immunocytochemical analysis of amygdaloid cells that immunoreact positively for Met-enkephalin. The results indicated the presence of many retrogradely labelled and immunocytochemically positive cells within the CE and adjoining regions of the lateral nucleus as well as others that were double labelled. The overall findings support the view that the CE powerfully inhibits affective defense behavior at the level of the PAG and that this phenomenon is mediated via an enkephalinergic mechanism.

Amygdaloid kindling during wakefulness and paradoxical sleep in the cat. 2. Sleep organization changes produced by kindling development

The effect of daily electrical amygdaloid stimulation (kindling, K) during waking (W-K) and paradoxical sleep (PS-K) on sleep organization was tested in 2 groups of chronically implanted adult male cats. Animals were stimulated 3 h after the 8-h sleep recordings were begun. Percentage, total time, and mean duration and number of episodes of W, slow wave sleep I and II, and PS stages were determined. Also, the mean interval of occurrence and hourly accumulation of PS were assessed. Recordings were performed before, throughout, and immediately after kindling and 2 months after the last recorded kindled seizure. Analysis of total recording time demonstrated that in both W-K and PS-K animals the kindling process provoked only transitory changes which returned to baseline values during kindling and immediately after. Analysis of pre- and post-stimulation periods demonstrated compensatory changes in W and PS percentage during the late stages of kindling. We conclude that rather than sleep diminution, kindling provokes a circadian shift of W and PS stages.

Interactions between amygdaloid and hypothalamic self-stimulation: a re-examination

The function relating bar-pressing rate to the frequency of cathodal pulses was obtained in rats self-stimulating with amygdaloid (AMY) and lateral hypothalamic (LH) electrodes. The maximum self-stimulation (SS) rates in the AMY was found to be very low, compared to the LH. Concurrent stimulation with pairs of AMY-LH pulses did not shift the rate-frequency functional laterally, indicating the absence of summation of the two rewarding effects. In a second experiment, concurrent AMY-LH stimulation (using sub-threshold intensity LH pulses) facilitated bar-pressing for AMY stimulation (it increased the slope of the AMY rate-frequency function) without shifting this function laterally. In a third experiment, subjects were given a choice between a pulse frequency yielding maximal AMY rate and a series of higher pulse frequencies. Subjects consistently preferred the higher frequency values, attesting that the maximum AMY rates were not constrained by a saturating reinforcing effect. In a fourth experiment, subjects were given a choice between AMY stimulation and concurrent AMY-LH stimulation, using low intensity LH pulses. Subjects showed no preference for either stimulation condition, although rates were higher for the latter condition. These findings suggest that the maximum rate for AMY stimulation was constrained by factors interfering with bar-pressing and that the effect of these factors was attenuated by co-activation of the LH. In a fifth experiment, pre-treatment with phenobarbital mimicked the rate-enhancing effect of concurrent AMY-LH stimulation for 2 of the 4 subjects tested. This finding suggests that the LH pulses contributed to attenuate seizure activity accompanying AMY SS. In a final experiment, AMY SS rates were also increased by co-activation of rewarding sites in the rostral MFB but not the dorsal raphe, suggesting an anatomical specificity of this effect.