Neuropeptide Y receptor subtypes in the basolateral nucleus of the amygdala modulate anxiogenic responses in rats

The behavioral effects induced by intra-amygdala stimulation of the neuropeptide Y (NPY) Y(2) and the NPY Y(5) receptor subtypes were assessed in the social interaction (SI) test. Microinjections of NPY(3-36), an NPY Y(2) preferring agonist, into the basolateral nucleus of the amygdala (BLA) produced bi-directional dose-response curve. At low doses NPY(3-36) has an anxiogenic effect while at higher doses it produced an anxiolytic effect. Pretreatment with the NPY Y(5) receptor antagonist Novartis 1(1 nmol), an analog of CGP71683A synthesized by Eli Lilly and Company, IN, blocked the anxiolytic effects of NPY(3-36) (80 pmol), while pretreatment with BIBO 3304 (200 pmol), a Y(1) antagonist, had no effect, suggesting that the Y(5), but not the Y(1) receptor was involved in the anxiolytic behavior produced following intra-amygdalar NPY(3-36) administration. In addition, the Y(5) antagonist had no behavioral effect when given alone at 1.0 nmol. These findings support the hypothesis that amygdalar Y(2) receptors may play a role in mediating anxiogenic effects, while Y(5) receptors may be involved in the anxiolytic behaviors of NPY.     

Analgesia and hyperractivity following morphine microinjection into mouse brain.

Analgesia and a paradoxical hyperreactivity to stimuli of sudden onset have recently been reported following the microinjection of morphine into the periacqueductal gray matter of rats. These effects have not been systematically investigated in other species. In the present study, both analgesia and hyperreactivity were observed as dose dependent effects of morphine microinjection into the periacqueductal gray matter of several strains of mice. Analgesia alone was produced by low doses of morphine while at higher doses analgesia was accompanied by hyperreactivity. Strain differences were noted with B6D2F1 mice being more susceptible to the hyperreactivity following morphine than BALB/c mice.     

Elevation of aversive threshold in rats by intra-amygdaloid injection of morphine sulphate

Bilateral micro-injection of morphine sulphate (10μg, 20μg) into the cortico-medial amygdala produced a dose-dependent increase in aversive threshold. Similar injections into the basolateral amygdala or caudate-putamen failed to have any consistent effect on aversive thresholds. Whilst overall activity levels remained unaffected by morphine injection into either amygdaloid site, caudate animals exhibited a significant decrement in total activity in response to both morphine and control injections. Results are discussed with reference to a possible role for limbic mechanisms in morphine analgesia.     

Reinforcing effects of morphine microinjection into the ventral tegmental area

A neural substrate for the reinforcing property of an opiate drug was identified in the ventral tegmental area (VTA) by establishing conditioned reinforcement to salient environmental stimuli paired with intracerebral microinjections of morphine. Bilateral microinjections of morphine into the VTA in doses of 0.2 μg and 1.0 μg produced a subsequent change in place preference to a distinctive compartment previously associated with the stimulant effects of morphine. Microinjection of 1.0 μg morphine at sites 2.5 mm dorsal to the VTA had no effect. Pretreatment with naloxone (2 mg/kg) antagonized the reinforcing effects of 1.0 μg morphine as this group showed no significant change in place preference. Nor did control groups receiving microinjections of sterile physiological saline. Taken together, these data suggest that opiate receptors, located in the ventral tegmental area, play an important role in mediating the reinforcing effects of morphine. The possible involvement of dopaminergic neurons in these effects is discussed.     

Differential behavioral effects following microinjection of an NMDA antagonist into nucleus accumbens subregions

Recent studies have demonstrated the existence of two distinct regions within the nucleus accumbens (N.Acc) known as core and shell. In order to investigate whether the behavioral functions of excitatory amino acid receptors differed between these two subregions, rats were administered microinjections of 2-amino-5-phosphonovaleric acid (AP-5), a competitive NMDA antagonist (0, 0.05, 0.2, 0.5, 1.0 µg/0.5 µl) into selected central and medial regions of the accumbens. The central and medial sites were assumed to correspond approximately to core and shell subregions, respectively. The animals were tested in two exploratory tasks: the open field and a novel object test. In the open field test, AP-5 significantly decreased peripheral locomotion and center rearing frequency in the central but not the medial group. Locomotion and rearing were not affected by AP5 infusion into a control site, the anterior dorsal striatum (ADS). In the novel object test, animals were tested in the same open field, with prior habituation, and with several novel objects placed within it. In this test, infusions of AP-5 (0, 1.0 µg/0.5 µl) decreased the number and duration of contacts with the novel objects in the central but not the medial group. In addition, peripheral and center locomotion were decreased by AP-5 infusions into the central site, whether objects were present or not. In contrast, AP-5 infusions into the medial site elicited an increase in peripheral locomotion in both stimulus conditions. These findings provide behavioral-pharmacological evidence that the central and medial subregions of the nucleus accumbens can be differentiated. Moreover, the results suggest that exploratory motor responses may be dependent on glutamate-coded input to the nucleus accumbens area corresponding to the core region.     

Corticotropin-releasing factor microinjection into the central nucleus of the amygdala alters REM sleep

Psychological stressors have a prominent effect on rapid eye movement sleep (REMS) in humans and animals. We hypothesized that the stress-related neurochemical corticotropin-releasing factor (CRF), acting in the amygdala, could initiate neural events that lead to REMS alterations. Therefore, we made bilateral microinjections of three different doses of CRF into the central nucleus of the amygdala (CeA) in five rats. Only the lowest dose of CRF (1 ng) induced a change in sleep, specifically REMS, during the 4-h post-injection period. Thus, REMS alterations following psychological stress may depend, in part, on CRF release in the CeA.     

Involvement of noradrenergic system within the central nucleus of the amygdala in naloxone-precipitated morphine withdrawal-induced conditioned place aversion in rats

Rationale We previously reported that the central nucleus of the amygdala (CeA) plays a crucial role in the negative affective, rather than somatic, component of morphine withdrawal. However, numerous studies have reported that the central ascending noradrenergic system is implicated in morphine withdrawal syndrome, although the roles of the noradrenergic system within the CeA in the negative affective component remains less clear. Objectives The possible role of the noradrenergic system within the CeA in the negative affective component of morphine withdrawal was investigated. Methods The extracellular noradrenaline level within the CeA during naloxone-precipitated morphine withdrawal was measured using an in vivo microdialysis experiment on unanesthetized and freely moving rats. The effects of microinjection of β-adrenoceptor antagonists into the bilateral CeA on the naloxone-precipitated morphine withdrawal-induced conditioned place aversion (CPA) and somatic signs were examined. Results The extracellular noradrenaline level within the CeA was transiently elevated during morphine withdrawal. Intra-CeA injections of β-adrenoceptor antagonists propranolol (30 nmol per side) and timolol (10 nmol per side) significantly attenuated the morphine withdrawal-induced CPA. Similarly, β₁-antagonist atenolol (30 nmol per side) or β₂-antagonist butoxamine (30 nmol per side) significantly attenuated the CPA. In contrast, they did not affect morphine withdrawal-induced somatic signs, except for propranolol. Conclusion These results suggest that the activation of the noradrenergic system within the CeA contributes to naloxone-precipitated morphine withdrawal-induced CPA, rather than somatic signs, through β₁- and β₂-adrenoceptors.     

Adult behavior and adrenocortical function following neonatal morphine treatment in rats

Adult behavioral and endocrine effects of neonatal administration of morphine (M) were studied in female rats injected s.c. with M on either days 3–12 (M₁) or days 12–21 (M₂). The dose given twice daily was increased to 8 mg/kg in group M₁ and to 16 mg/kg in group M₂. Compared to saline-treated controls, growth rates were temporarily suppressed (P<0.05) and body weights were reduced (P<0.05) util day 20 in M₁ and until day 64 in M₂ rats. The open-field test performed on days 29–31 failed to differentiate between neonatal treatment groups. On days 90–95 M₁ but not M₂ animals showed impaired learning of a conditioned emotional responses (CER). On day 40 all groups showed similar increased levels of plasma corticosterone 30 min following injection of naloxone (5 mg/kg). Compared to controls on day 156, both M₁ and M₂ rats showed diminished (P<0.05) analgesic responses (hot-plate test) to M (10 mg/kg). In response to M challenge (40 mg/kg) on day 170, all groups showed comparable acute increases in plasma corticosterone levels. These findings provide further evidence that early exposure to M results in growth retardation and protracted tolerance and that, depending on dose and time of exposure, neonatal M may result in impaired learning of CER in adulthood. These effects suggest that early morphine can produce long-lasting alterations of learned behavior without lasting impairment of pituitary-adrenal function.     

Neuropeptide Y in the central nucleus of amygdala regulates the anxiolytic effect of agmatine in rats

In the present study, modulation of anxiolytic action of agmatine by neuropeptide Y (NPY) in the central nucleus of amygdala (CeA) is evaluated employing Vogel's conflict test (VCT) in rats. The intra-CeA administration of agmatine (0.6 and 1.2 µmol/rat), NPY (10 and 20 pmol/rat) or NPY Y1/Y5 receptors agonist [Leu³¹, Pro³⁴]-NPY (30 and 60 pmol/rat) significantly increased the number of punished drinking licks following 15 min of treatment. Combination treatment of subeffective dose of NPY (5 pmol/rat) or [Leu³¹, Pro³⁴]-NPY (15 pmol/rat) and agmatine (0.3 µmol/rat) produced synergistic anxiolytic-like effect. However, intra-CeA administration of selective NPY Y1 receptor antagonist, BIBP3226 (0.25 and 0.5 mmol/rat) produced anxiogenic effect. In separate set of experiment, pretreatment with BIBP3226 (0.12 mmol/rat) reversed the anxiolytic effect of agmatine (0.6 µmol/rat). Furthermore, we evaluated the effect of intraperitoneal injection of agmatine (40 mg/kg) on NPY-immunoreactivity in the nucleus accumbens shell (AcbSh), lateral part of bed nucleus of stria terminalis (BNSTl) and CeA. While agmatine treatment significantly decreased the fibers density in BNSTl, increase was noticed in AcbSh. In addition, agmatine reduced NPY-immunoreactive cells in the AcbSh and CeA. Immunohistochemical data suggest the enhanced transmission of NPY from the AcbSh and CeA. Taken together, this study suggests that agmatine produced anxiolytic effect which might be regulated via modulation of NPYergic system particularly in the CeA.     

Dependence to morphine in differentially housed rats

Although the effects of differential housing, particularly isolation, on the action of several classes of pharmacological agents have been studied, little attention has been given to this factor in regard to narcotics. The present study involves the effects of long-term social isolation on dependence to morphine produced by pellet implants in rats. When abstinence was precipitated with naloxone, isolated rats demonstrated less jumping and less diarrhea than grouped rats. No differences were found in other signs. In addition, the differences were seen both in isolates developing muricidal behavior and those not developing this behavioral pattern.Supported in part by G. L. Pfeiffer Foundation, N.Y., NATO Research Grant No. 719, USPHS Grants DA 00049 and DA 00376.Visiting Scientist from the Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pa., U.S.A.     

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in sprague-dawley rats

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg⁶ levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg⁶ levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg⁶ and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg⁶ levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg⁶ levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointer-mediate lobe were even lower in the withdrawal state. In conclusion, morphine tolerance and withdrawal affected prodynorphin-derived peptides in areas related to central reward mechanisms, and in the pituitary gland. The dynorphin peptides and the LeuenkephalinArg⁶ fragment were not affected similarly, indicating an effect also on metabolic interconversion.     

Immediate-early gene expression in the central nucleus of the amygdala is not specific for anxiolytic or anxiogenic drugs

The lateral, basal, and central nuclei of the amygdala are part of a circuitry that instantiates many fear and anxious behaviors. One line of support indicates that immediate-early gene (IEG) expression (e.g., c-fos and egr-1 (zif268)) is increased in these nuclei following fear conditioning. Other research finds that anxiogenic drugs working through various mechanisms induce IEG expression in the central nucleus of the amygdala (CeA) suggesting that expression is a neural marker for fear and anxiety. However, several studies have also found that anxiolytic drugs induce IEG expression in the CeA. Expression of egr-1 in the CeA and lateral nucleus of the amygdala following administration of anxiolytic and anxiogenic benzodiazepine and serotonin agonists and antagonists was investigated. The first experiment determined behaviorally active anxiolytic and anxiogenic doses for two anxiogenic drugs (FG 7142 and mCPP) and two anxiolytic drugs (diazepam and buspirone). The effects of anxiogenic and anxiolytic doses of these drugs on egr-1 expression in the amygdala were then tested in a second experiment. All four drugs increased egr-1 in the CeA indicating that increased egr-1 mRNA expression in the CeA is not specific to anxiolytic or anxiogenic effects of the drugs. We suggest that IEG expression in the CeA may be due to activation of circuits that are associated with systemic physiological homeostasis perturbed by a number of drugs including anxiogenic and anxiolytic compounds.     

A role for the CRF-containing pathway from central nucleus of the amygdala to bed nucleus of the stria terminalis in the stress-induced reinstatement of cocaine seeking in rats

RATIONALE: We reported previously that bilateral injection of a corticotropin-releasing factor (CRF)-receptor antagonist, D-Phe CRF(12-41), into the bed nucleus of the stria terminalis (BNST) blocks the reinstatement of cocaine seeking induced by footshock, whereas the injection of CRF into the same region induces reinstatement. One source of CRF in the BNST arises from a CRF-containing projection originating in the central nucleus of the amygdala (CeA). OBJECTIVE: To determine whether the CRF-containing projection from the amygdala to the BNST is involved in the mediation of stress-induced reinstatement of cocaine seeking by functionally interrupting the pathway. METHODS: Rats trained to self-administer cocaine (1 mg/kg, IV, 9 days) were given extinction sessions after a 10- to 11-day drug-free period, followed by tests for stress-induced reinstatement (footshock: 15 min intermittent 0.8-mA footshocks given immediately before presentation of the previously active lever). Before the tests, animals were pretreated with either: (1) TTX (2.5 ng) in amygdala (including the CeA) in one hemisphere and D-Phe CRF(12-41) (50 ng) in BNST in the other, (2) unilateral TTX, or (3) unilateral D-Phe. RESULTS: Footshock reinstated cocaine seeking following unilateral injections of either TTX in amygdala or D-Phe in BNST, but following the injection of both TTX in amygdala and D-Phe in BNST the effects of footshock were greatly attenuated. CONCLUSION: These results suggest that the CRF-containing pathway from CeA to BNST is involved in mediating the effects of CRF and its receptor antagonist in the BNST on the reinstatement of cocaine seeking.     

Involvement of caudate nucleus,amygdala or reticular formation in neuroleptic and narcotic catalepsy

Local injection of haloperidol into the caudate nucleus produced catalepsy in contrast to the weak effects of morphine injected at the same site. Injection of either haloperidol or morphine into the amygdala did not have any cataleptogenic effect. Both haloperidol and morphine produced catalepsy when injected into the reticular formation. Naloxone injected into the reticular formation completely reversed the catalepsy following intraperitoneal morphine but not haloperidol. Pretreatment with α-methyl-p-tyrosine potentiated the effect of haloperidol injected into either the reticular formation or caudate nucleus. Phentolamine but not lignocaine or metergoline, injected into the reticular formation also caused a cataleptic response. The results confirm the caudate nucleus as a site for haloperidol catalepsy and in addition, suggest the reticular formation as a primary site for morphine catalepsy and a secondary site for haloperidol catalepsy; additionally a noradrenergic modulation of catalepsy may occur within this brainstem region.     

Opiate effects in the amygdala central nucleus on heart rate conditioning in rabbits

Opiate agents were administered into the central nucleus of the amygdala complex of rabbits prior to either classical conditioning or pseudoconditioning of heart rate responding. Compared to control groups, opiate administration into the central nucleus did not significantly alter baseline heart rate, heart rate responding during habituation trials to presentations of the conditioned stimulus alone, or heart rate responding during the pseudoconditioning procedure. However, opiate administration altered the acquisition of a conditioned bradycardia response during classical conditioning trials in which the offset of the conditioned stimulus was coincident with the presentation of an aversive unconditioned stimulus. The opiate agonist levorphanol (5.0 nmole) significantly impaired the acquisition of the conditioned bradycardia response. This effect was observed to be stereospecific and blocked by concurrent administration of the opiate antagonist naloxone (2.5 nmole). Naloxone (2.5 nmole) administration alone significantly increased the magnitude of the conditioned bradycardia response. These effects produced by opiate administration into the central nucleus were not observed following administration of the same agents into sites 1–2 mm dorsal to the central nucleus.     

Changes in morphine self-administration and morphine dependence after lesions of the caudate nucleus in rats

Rats were trained to bar press for intravenous infusions of morphine sulfate during 1-hr daily test sessions. Small, centrally placed bilateral lesions of the caudate nucleus reduced rates of morphine self-administration to approximately one seventh of preoperative levels; postoperative rates were similar to preoperative rates when the postoperative unit infusion dose of morphine was one tenth of the preoperative dose. Caudate lesions also lowered the threshold dose at which morphine's rewarding property could be detected. Physical dependence was studied in other rats receiving a 3-day continuous infusion of morphine sulfate via implanted subcutaneous silicone reservoirs. Caudate lesions ameliorated withdrawal-induced weight loss and naloxone-induced wet dog shakes. Both the self-administration and dependence data are consistent with the idea that morphine blocks dopaminergic transmission in the striatum.     

Role of catecholaminergic mechanisms in the expression of the morphine abstinence syndrome in rats

The effect of various drugs affecting catecholaminergic mechanisms on the precipitated morphine withdrawal syndrome was studied in rats which had developed a medium degree of dependence. Administration of low doses of d-amphetamine, cocaine, and L-Dopa shortly before precipitating withdrawal by levallorphan induced a dose-dependent increase of dominant withdrawal signs such as jumping and a decrease of recessive signs such as wet dog shaking; signs such as diarrhea and ptosis decreased, whereas rhinorrhea, salivation and lacrimation increased. A qualitatively very similar change in withdrawal signs occurred when withdrawal was precipitated in extremely highly dependent rats and/or increasing doses of the antagonist were administered. Therefore, the effects of the above drugs are interpreted as potentiation of withdrawal. Pretreatment with higher doses of the same drugs provoked strong stereotyped behaviour which obviously suppressed the occurrence of other motor signs.Activation of noradrenergic or dopaminergic mechanisms with desipramine or apomorphine induced an increase in the intensity of withdrawal, which was, however, much more pronounced after the former than the latter drug. When catecholamines (CA) were previously depleted by alpha-methyl-para-tyrosine (AMT), apomorphine lost a great part of its effectiveness. Blockade of CA synthesis by AMT alone resulted in decreased jumping while at the same time writhing largely increased, thus, inducing a profile of signs characteristic for a weak withdrawal. Selective inhibition of noradrenaline synthesis by FLA-63 resulted in a reduction in withdrawal intensity. Ro 4-4602 + L-Dopa, given after AMT, antagonized and reversed the reduction of withdrawal, but this effect was not so pronounced when by additional pretreatment with FLA-63 NA levels remained low. It is concluded that of both brain CA especially noradrenaline is involved in the manifestation of the morphine withdrawal syndrome.     

Enkephalin analogue effects in the amygdala central nucleus on conditioned heart rate

The experiment was conducted to assess the effects of enkephalin analogue administration into the amygdala central nucleus on the acquisition of classically conditioned heart rate responding in rabbits. Bilateral injections of either D-Ala2, Met5-enkephalinamide (DALA) or D-Ala2, D-Leu5-enkephalin (DADL) were administered in a 1.0 microliter volume into the central nucleus immediately prior to the conditioning session. Administration of DALA significantly attenuated the acquisition of conditioned heart rate responding whereas groups which received comparable doses of DADL exhibited conditioned responding which did not differ from the vehicle-injected control group. The effect on conditioned responding produced by DALA administration was blocked by concurrent administration of the opiate antagonist naloxone. These results provide some support for the involvement of mu receptor activity within the central nucleus region of the amygdala in conditioning processes.     

GABAA receptors are involved in the analgesic effects of morphine microinjected into the central nucleus of the amygdala

The central nucleus of the amygdala (CeA) has an important role in pain perception and analgesia. Opioid and GABA_A receptors, which are both involved in pain modulation, are found in high concentration in the CeA. The present study was designed to examine the interaction of opioidergic and GABAergic systems in the CeA during modulation of acute thermal pain. In the present study, different doses of morphine (25, 50 and 100 μg/rat), either alone or after 5 min pretreatment with the selective GABA_A receptor agonist muscimol (60 ng/rat) or the selective GABA_A receptor antagonist bicuculline (50 ng/rat), were injected bilaterally into the CeA of each rat. Tail‐flick latencies (TFL) were measured every 5 min for 60 min. The results revealed that microinjection of morphine into the CeA significantly increased TFL in a dose‐dependent manner. Microinjection of bicuculline or muscimol in combination with morphine into the CeA increased and decreased TFL, respectively. It seems that morphine in the CeA facilitates the function of descending inhibitory systems by interacting with the activity of local GABA_A receptors.