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Evidence for the presynaptic localization of opiate binding sites on striatal efferent fibers
Abstract
Using quantitative receptor autoradiography, [3H]D-Ala-D-Leu-enkephalin (DADL) and [3H]naloxone binding were studied in rat striatum and striatal projection areas (globus pallidus (GP) and substantia nigra pars reticulata (SNr] after unilateral striatal kainic acid lesions. [3H]DADL and [3H]naloxone binding were each examined by two methods. Initially, [3H]DADL binding was performed in 50 mM Tris-HCl (pH 7.4), 30 mM NaCl, 3 mM manganese acetate and 2 microM GTP; [3H]naloxone binding was carried out in 50 mM Tris-HCl (pH 7.4) and 100 mM NaCl. Subsequent studies were carried out in 150 mM Tris-HCl (pH 7.4) and either [3H]DADL plus 500 nM morphiceptin (to block [3H]DADL binding to mu receptors) or [3H]naloxone plus 10 nM delta receptor peptide (to block [3H]naloxone binding to delta receptors). At one and eight weeks in the lesioned striatum, [3H]DADL binding was reduced by 70% and 82%, respectively, when compared to the control side. [3H]Naloxone binding was reduced by 35% and 20%. In GP and SNr, [3H]DADL binding was reduced by 31% and 41%, respectively, at one week and 27% and 26% at eight weeks. [3H]Naloxone binding was reduced 19% in GP at eight weeks. A parsimonious explanation of these results is that opiate binding sites are located on presynaptic terminals of striatal efferent fibers to globus pallidus and substantia nigra pars reticulata as well as on local striatal axon collaterals. Since opiate peptides have recently been found to coexist with GABA in some striatal neurons, opiate peptides may play a role in striatal function by controlling GABA release from striatal efferent fibers. It is possible that pallidal and nigral opiate binding could be utilized as a marker for striatal terminals.
... It has been shown that striatal neurons may contain both GABA and opiatelike immunoreactivity. 37 This has lead to the speculation that opiates may block GABA release and modulate the basal ganglia outflow pathway. ...
... 38 It has been proposed that striatal mu and lambda receptors are partially on the presynaptic axons of nigral dopaminergic neurons. 39 In the pallidum and substantia nigra pars reticulata, lesion studies suggest that some of the mu and lambda sites are on presynaptic terminals of striatofugal neurons, 37 minergic neurons. 14 Kappa sites are located in the substantia nigra pars reticulata in rats. ...
... (Figure 3) In an animal model of Huntington's disease, Abou-Khalil et al have found a reduced binding for the lambda site in pallidum and substantia nigra pars reticulata. 37 In Parkinson's disease, both a reduction in striatal lambda binding 40 and an increase in this class of receptors has been reported. 18 In the substantia nigra in patients with this condition, a reduction in kappa and mu binding has been found. ...
The study of neurotransmitter receptors aids in the understanding of the normal anatomy, pharmacology, therapeutics and pathophysiology of disease processes involving the basal ganglia. Receptors may be studied in vitro by homogenate binding experiments, enzyme analysis or quantitative autoradiography and in vivo with positron emission tomography. In the substantia nigra (SN), receptors have been identified for somatostatin, neurotensin, substance P, glycine, benzodiazepine and GABA, opiates, dopamine, angiotensin converting enzyme (ACE) and serotonin. The striatum has receptors for dopamine, GABA and benzodiazepines, acetylcholine, opiates, substance P, glutamate and cholecystokinin. GABA and benzodiazepine receptors are also located in the globus pallidus. In Parkinson's disease, striatal dopamine D-2 receptors are elevated in patients that have not received L-DOPA therapy. This supersensitivity is reversed with agonist therapy. Muscarinic binding to cholinergic receptors seems to correlate with dopamine receptors. Delta opiate receptors are increased in the caudate and mu binding is reduced in the striatum. In the SN of patients with Parkinson's disease, there is reduced binding of somatostatin, neurotensin, mu and kappa opiates, benzodiazepine and GABA and glycine. In Huntington's disease, there is reduced binding of GABA and benzodiazepines, dopamine, acetylcholine, glutamate and CCK. There is increased binding of GABA in both the SN and globus pallidus. Glycine binding is increased in the substantia nigra and ACE is reduced.
... There is independent evidence for the existence of opioid receptors on the striatonigral cells. Lesions of the striatum with kainic acid injection results in reductions of numbers of striatal binding sites for various opioid receptor ligands by 38-70% ( Pollard et al., 1978;Abou-Khalil et al., 1984;Van der Kooy et al., 1986;Eghbali et al., 1987). At least some of this (1 and 6) binding appears to be on cells projecting to the substantia nigra because opioid receptor binding in the nigra is also reduced following striatal lesions ( Abou-Khalil et al., 1984). ...
... Lesions of the striatum with kainic acid injection results in reductions of numbers of striatal binding sites for various opioid receptor ligands by 38-70% ( Pollard et al., 1978;Abou-Khalil et al., 1984;Van der Kooy et al., 1986;Eghbali et al., 1987). At least some of this (1 and 6) binding appears to be on cells projecting to the substantia nigra because opioid receptor binding in the nigra is also reduced following striatal lesions ( Abou-Khalil et al., 1984). Furthermore, section of the nigrostriatal pathway reduces numbers of opioid binding sites in the nigra ( Gale et al., 1979) and [3H]etorphine binding accumulates rostra1 to a nigrostriatal pathway transsection (Van der Kooy et al., 1986). ...
Intracellular recordings were made from 475 rat substantia nigra zona compacta neurons in vitro. The region from which recordings were made was rich in catecholamine fluorescence. Two groups of neuron, termed principal neurons (95% of the total) and secondary neurons (5% of the total) were clearly distinguishable according to one or more of the following 4 electrophysiological properties. Secondary neurons (23 cells) (1) fired spontaneous action potentials at frequencies greater than 10 Hz, or were quiescent (30%); (2) had action potentials less than 1 msec in duration; (3) did not show time-dependent inward rectification with step hyperpolarization; and (4) had slope conductances of about 4 nS (between -75 and -90 mV). In contrast, principal neurons (1) fired spontaneous action potentials in the range 1-8 Hz, or were quiescent (33%); (2) had action potentials greater than 1 msec in duration; (3) showed pronounced time-dependent inward rectification; and (4) had steady-state membrane slope conductances of around 22 nS (between -75 and -90 mV). Secondary cells were not affected by dopamine but were hyperpolarized by baclofen, GABA, and the mu opioid receptor agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO). On the other hand, dopamine and baclofen inhibited firing and/or hyperpolarized all principal cells tested, but mu or delta opioid receptor agonists had no effect. The properties of these 2 cell types broadly correspond with those described by electrophysiological studies in vivo, in which case the majority, or principal, cells are believed to be dopaminergic.(ABSTRACT TRUNCATED AT 250 WORDS)
... [3H]Spiroperidol binding was performed as described previously by Pan et al. (1984n) Abou-Khalil et al. (1984). ...
... BDZ receptors also up-regulate after striatal lesions (Shibuya et al., 1980;Biggio et al., 1981;Lo et al., 1983;Pan et al., 1984~); however, the time course of BDZ receptor changes is slower than that for GABA receptors (Pan et al., 1984~). In contrast, opiate receptors (both p and S) decrease in striaturn and its projection areas after striatal lesions (Abou-Khalil et al., 1984). Although no direct proof is available, these findings are consistent with the postsynaptic localization and up-regulation of GABA and BDZ receptors and the presynaptic (terminal) localization of opiate receptors. ...
Quantitative autoradiography was used to ascertain alterations in [3H]muscimol, [3H]flunitrazepam (FLU), [3H]naloxone, [3H]D-alanine-D-leucine-enkephalin (DADL), and [3H]spiroperidol binding in basal ganglia 1 week, 4 weeks, and 5 months after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle (MFB) in the rat. At 1 and 4 weeks following lesions, [3H]spiroperidol binding increased 33% in striatum. At 5 months, [3H]spiroperidol was only nonsignificantly increased above control. At 1 week, [3H]muscimol binding decreased 39% in ipsilateral globus pallidus (GP), but increased 41% and 11% in entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr), respectively. At 4 weeks, [3H]muscimol binding was reduced 19% in striatum and 44% in GP and remained enhanced by 32% in both EPN and SNr. These changes in [3H]muscimol binding persisted at 5 months. [3H]FLU binding was altered in the same direction as [3H]muscimol binding; however, changes were slower in onset and became significant (and remained so) only at 4 weeks after lesions. Decreases in [3H]naloxone and [3H]DADL binding were seen in striatum, GP, EPN, and SNr. Scatchard analyses revealed that only receptor numbers were altered. This study provides biochemical evidence for differential regulation of striatal GABAergic output to GP and EPN/SNr.
... The few large polygonal neurons which were observed were also unlabeled (Fig. 3E). To investigate further the apparent clustering of labeled and unlabeled neurons, in situ hybridization for mGluR5 was performed in coronally cut adult rat brain sections with serially adjacent sections processed for )H-naloxone binding by the method of Abou-Khalil et al. (1984) to define striosome and matrix compartments. No simple relationship between neurons labeled with probes for mGluR5 and the compartments defined by 3H-naloxone binding was observed, and clusters of labeled and unlabeled neurons were present in both compartments. ...
Metabotropic glutamate receptors (mGluRs) couple the actions of glutamate to intracellular second messenger systems through G-proteins. The mGluRs play an important role in the regulation of basal ganglia function. Ligand binding studies have revealed that the basal ganglia contain at least two pharmacological types of metabotropic binding sites. Agonists of mGluRs can affect both in vitro electrophysiologic responses of striatal neurons and motor behavior in vivo. Recently, cDNAs encoding five mGluRs have been cloned, each with distinct structural and pharmacological properties. In order to elucidate the function of these receptors in the biology of the extrapyramidal motor system, we have used in situ hybridization to examine the regional and cellular expression patterns of mGluR1-mGluR5 in the adult rat basal ganglia. In the striatum, all of these mGluRs were present in widely varying relative densities and cellular patterns. MGluR5 was particularly prominent, and exhibited a heterogeneous cellular distribution, with labeled and unlabeled populations of neurons. MGluR2 was expressed in a small population of large polygonal striatal neurons. The subthalamic nucleus was the only other basal ganglia structure that expressed mGluR2. Distinct cellular distributions of mGluR expression were also observed within the nucleus accumbens, globus pallidus, ventral pallidum, and substantia nigra pars reticulata. MGluR3 was expressed in glia in all basal ganglia structures, but was observed in neurons only in the striatum, substantia nigra pars reticulata, and very weakly in the subthalamic nucleus. Comparison of the restricted mGluR2 and mGluR3 mRNA distributions with that of metabotropic ligand binding sites supports a possible presynaptic location for these receptors in the basal ganglia. MGluR1 was the only mGluR message prominently expressed in the dopaminergic neurons of the substantia nigra pars compacta, suggesting the involvement of this receptor in the regulation of dopamine release from nigrostriatal terminals.
... GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids. It is known that opioids interact at GABAergic neurons and axon terminals and that such interaction produces neuronal inhibition comparable, perhaps, to that reported in the striatonigral and nigrostriatal systems of the rat (Iwatsubo and Kondo, 1978;Turski et al., 1982;Abou-Khalil et al., 1984;Starr, 1985). The extraordinarily high levels of mu-opioid receptors in the rat AOS nuclei, and the opioid effects on visual sensitivity (Rothenberg et al., 1979) diminished gain of smooth pursuit and saccadic velocities (Rothenberg et al., 1980a, b), suggests that endogenous opiates may play a role in regulating visual transmission through the AOS nuclei, and through connections with the vestibular nuclei and precerebellar pathways, may account, in part, for such phenomena as the fluctuating gain of optokinetic nystagmus and other visuomotor reflexes. ...
... GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids. It is known that opioids interact at GABAergic neurons and axon terminals and that such interaction produces neuronal inhibition comparable, perhaps, to that reported in the striatonigral and nigrostriatal systems of the rat (Iwatsubo and Kondo, 1978;Turski et al., 1982;Abou-Khalil et al., 1984;Starr, 1985). The extraordinarily high levels of mu-opioid receptors in the rat AOS nuclei, and the opioid effects on visual sensitivity (Rothenberg et al., 1979) diminished gain of smooth pursuit and saccadic velocities (Rothenberg et al., 1980a, b), suggests that endogenous opiates may play a role in regulating visual transmission through the AOS nuclei, and through connections with the vestibular nuclei and precerebellar pathways, may account, in part, for such phenomena as the fluctuating gain of optokinetic nystagmus and other visuomotor reflexes. ...
... GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids. It is known that opioids interact at GABAergic neurons and axon terminals and that such interaction produces neuronal inhibition comparable, perhaps, to that reported in the striatonigral and nigrostriatal systems of the rat (Iwatsubo and Kondo, 1978;Turski et al., 1982;Abou-Khalil et al., 1984;Starr, 1985). The extraordinarily high levels of mu-opioid receptors in the rat AOS nuclei, and the opioid effects on visual sensitivity (Rothenberg et al., 1979) diminished gain of smooth pursuit and saccadic velocities (Rothenberg et al., 1980a, b), suggests that endogenous opiates may play a role in regulating visual transmission through the AOS nuclei, and through connections with the vestibular nuclei and precerebellar pathways, may account, in part, for such phenomena as the fluctuating gain of optokinetic nystagmus and other visuomotor reflexes. ...
... GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids. It is known that opioids interact at GABAergic neurons and axon terminals and that such interaction produces neuronal inhibition comparable, perhaps, to that reported in the striatonigral and nigrostriatal systems of the rat (Iwatsubo and Kondo, 1978;Turski et al., 1982;Abou-Khalil et al., 1984;Starr, 1985). The extraordinarily high levels of mu-opioid receptors in the rat AOS nuclei, and the opioid effects on visual sensitivity (Rothenberg et al., 1979) diminished gain of smooth pursuit and saccadic velocities (Rothenberg et al., 1980a, b), suggests that endogenous opiates may play a role in regulating visual transmission through the AOS nuclei, and through connections with the vestibular nuclei and precerebellar pathways, may account, in part, for such phenomena as the fluctuating gain of optokinetic nystagmus and other visuomotor reflexes. ...
... GABAergic and opioidergic systems are closely linked and activity of the same neuron may be regulated directly by both GABA and opioids. It is known that opioids interact at GABAergic neurons and axon terminals and that such interaction produces neuronal inhibition comparable, perhaps, to that reported in the striatonigral and nigrostriatal systems of the rat (Iwatsubo and Kondo, 1978;Turski et al., 1982;Abou-Khalil et al., 1984;Starr, 1985). The extraordinarily high levels of mu-opioid receptors in the rat AOS nuclei, and the opioid effects on visual sensitivity (Rothenberg et al., 1979) diminished gain of smooth pursuit and saccadic velocities (Rothenberg et al., 1980a, b), suggests that endogenous opiates may play a role in regulating visual transmission through the AOS nuclei, and through connections with the vestibular nuclei and precerebellar pathways, may account, in part, for such phenomena as the fluctuating gain of optokinetic nystagmus and other visuomotor reflexes. ...
... Endogenous opioid peptides (EOP) produce a myriad of physiological functions in the central nervous system by binding with different opioid receptors, including m , d , and k -opioid receptors (MOR, DOR, and KOR) (Wen et al. 2010b ) . The DOR and its endogenous ligands (e.g., enkephalins) are widely expressed in the basal ganglia, STN, striatum, GP, and SNr (Abou-Khalil et al. 1984 ;Aubert et al. 2007 ;Mansour et al. 1993 ) . DOR has been well recognized for its neuroprotection (Chao et al. 2008Ma et al. 2005 ;Yang et al. 2009 ;Zhang et al. 2000Zhang et al. , 2002Feng et al. 2009 ) . ...
Acupuncture is widely accepted as an alternative therapy for Parkinson’s disease (PD) in East Asia. The aim of this chapter is to review the current progress of the clinical and experimental acupuncture studies. A number of clinical studies in China and Korea proved that the efficacy of acupuncture lies in improving the motor impairments and complications of PD and reducing the long-term medical side effects; however, certain problems in the study design possibly exaggerated the efficacy of acupuncture and crippled the power of these evidences. More strictly designed clinical studies following the principles of randomization, control, double blindedness, and placebo are needed, to furnish powerful evidence in order to verify the efficacy of acupuncture. Several bench studies have tried to clarify the proposed mechanisms of acupuncture therapy like delaying the dopamine (DA) neuron depletion, improving the dopaminergic (DA-ergic) system in the PD state, neuroprotective effects, amelioration of the motor control network in the basal ganglia, and to attenuate the oxidative stress in PD state. The limited present-day bench studies still have not been able to provide sufficient evidence for revealing the secrets of acupuncture. At the end of this chapter, a new experimental strategy has been introduced. On combining the basis of the traditional acupuncture theory with the observation that the stimulation of the somatosensory pathways affects the cerebral DA level, a modified electroacupuncture method—the so-called superficial electrical stimulation (SES), is proposed. We hope that general PD researchers would accept the acupuncture therapy to a greater extent in the future.
... doi:10.1371/journal.pone.0075099.g007 presynaptic inhibition of GABA-mediated transmission in substantia nigra reticulata (SNr) [77,78] and be involved in the improvement of behavioral dysfunction, more especially abnormal clasping movement. Moreover, we may also suggest that in pENK-injected R6/2 mice, enkephalin-induced presynaptic activation of m, d and k opioid receptors on subthalamic nucleus (STN) efferent [8,79] by inhibition of excitatory neurotransmission in SNr might reduce the activity of SNr and could then contribute to disinhibition of thalamus and consequently to the improvement of behavioral symptoms in this group. ...
The reduction of pre-enkephalin (pENK) mRNA expression might be an early sign of striatal neuronal dysfunction in Huntington's disease (HD), due to mutated huntingtin protein. Indeed, striatopallidal (pENK-containing) neurodegeneration occurs at earlier stage of the disease, compare to the loss of striatonigral neurons. However, no data are available about the functional role of striatal pENK in HD. According to the neuroprotective properties of opioids that have been recognized recently, the objective of this study was to investigate whether striatal overexpression of pENK at early stage of HD can improve motor dysfunction, and/or reduce striatal neuronal loss in the R6/2 transgenic mouse model of HD. To achieve this goal recombinant adeno-associated-virus (rAAV2)-containing green fluorescence protein (GFP)-pENK was injected bilaterally in the striatum of R6/2 mice at 5 weeks old to overexpress opioid peptide pENK. Striatal injection of rAAV2-GFP was used as a control. Different behavioral tests were carried out before and/or after striatal injections of rAAV2. The animals were euthanized at 10 weeks old. Our results demonstrate that striatal overexpression of pENK had beneficial effects on behavioral symptoms of HD in R6/2 by: delaying the onset of decline in muscular force; reduction of clasping; improvement of fast motor activity, short-term memory and recognition; as well as normalization of anxiety-like behavior. The improvement of behavioral dysfunction in R6/2 mice having received rAAV2-GFP-pENK associated with upregulation of striatal pENK mRNA; the increased level of enkephalin peptide in the striatum, globus pallidus and substantia nigra; as well as the slight increase in the number of striatal neurons compared with other groups of R6/2. Accordingly, we suggest that at early stage of HD upregulation of striatal enkephalin might play a key role at attenuating illness symptoms.
... Striatal dynorphinergic neurons project to the substantia nigra (SN) and contact tyrosine hydroxylase (TH)-positive dopaminergic neurons (Pickel et al., 1993). Since opioid receptors are found on presynaptic terminals in striatum and on glutamatergic neurons projecting from the subthalamic nucleus to the globus pallidus (Abou-Khalil et al., 1984), the neuroanatomical distribution of dynorphins favors a opioid-mediated mechanism by which dynorphins can regulate dopamine (DA) release and/or function in the striatum. A recent study employing electron microscopy with dual labeling of prodynorphin and either D1R or TH in the rat nucleus accumbens showed that a majority (63%) of prodynorphin-containing dendrites colocalized with the D1R (Hara et al., 2006). ...
Dynorphins, endogenous peptide neurotransmitters expressed in the central nervous system, have been implicated in diverse pathophysiological processes, including excitotoxicity, chronic inflammation, traumatic injury, cognitive impairment, and motor dysfunction, with significant changes with aging or age-related disease processes. This has led to the hypothesis that the suppression of dynorphin expression would produce beneficial effects on learning and memory and motor function. To assess the phenotypic manifestations of chronic suppression of endogenous dynorphin, knockout (KO) mice lacking the coding exons of the gene encoding the prodynorphin (Pdyn) precursor protein, were tested in a series of behavioral, biochemical, and molecular biological studies. Moderately aged Pdyn KO perform comparatively better than similarly aged wild-type (WT) mice in the water maze task, although no Pdyn effect was seen among young adult mice. In addition, young adult Pdyn KO mice show mildly improved performance on a passive avoidance task. Minimal baseline differences were noted in spontaneous locomotor activity in an open-field assay, but Pdyn deletion produced a relative sparing of motor dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus, striatum, and frontal cortex of WT mice by quantitative radioimmunoassay. While aging produces progressive decline in Dyn B in striatum and frontal cortex, Dyn A shows an upward trend in frontal cortex without significant change in striatum. Systemic MPTP produces significant short-term elevations in dynorphin peptides that regress to below baseline by 7 days. HPLC analysis of striatal dopamine shows an age-dependent increase in basal dopamine levels in Pdyn KO mice, an effect that is abolished after MPTP. Western blotting experiments demonstrate that Pdyn deletion is associated with greater phosphorylation at the serine-40 site of tyrosine hydroxylase (TH) despite relatively less total TH immunoreactivity, suggesting a suppressive effect of dynorphins on dopamine synthesis. Microarray analysis of hippocampal tissue from young and aged WT and Pdyn KO mice reveals a number of functional groups of genes demonstrating altered expression. The results of this dissertation support a role of endogenous dynorphins in age-associated cognitive and motor dysfunction.
The globus pallidus in the basal ganglia plays an important role in movement regulation. Neuropeptides and endocannabinoids are neuronal signalling molecules that influence the functions of the whole brain. Endocannabinoids, enkephalin, substance P, neurotensin, orexin, somatostatin and pituitary adenylate cyclase-activating polypeptides are richly concentrated in the globus pallidus. Neuropeptides and endocannabinoids exert excitatory or inhibitory effects in the globus pallidus mainly by modulating GABAergic, glutamatergic and dopaminergic neurotransmission, as well as many ionic mechanisms. Pallidal neuropeptides and endocannabinoids are associated with the pathophysiology of a number of neurological disorders, such as Parkinson's disease, Huntington's disease, schizophrenia, and depression. The levels of neuropeptides and endocannabinoid and their receptors in the globus pallidus change in neurological diseases. It has been demonstrated that spontaneous firing activity of globus pallidus neurons is closely related to the manifestations of Parkinson's disease. Therefore, the neuropeptides and endocannabinoids in the globus pallidus may function as potential targets for treatment in some neurological diseases. In this review, we highlight the morphology and function of neuropeptides and endocannabinoids in the globus pallidus and their involvement in neurological diseases.
The effects of 1 mg of a s).nthetic analogue of leu-enkephalin (Dalargin) injected inhamusculax tl rice daily for 3 consecutive days were studied in 20 patients vrith a.lcohol withdrawa.l slaldrome. The results suggest that the therapeutic effects of Dalargin in alcohol withdrawal s1'ndrome are mainly connected v/ith the changes in the dopamin-, serotonin-, GABA-and opioid-ergic neurotnnsmitters systems.
The study of both presynaptic and postsynaptic markers of neurotransmitter systems in the basal ganglia has afforded a great deal of information about the function of specific neuronal pathways in health and disease. In Parkinson’s disease, the emphasis has been on the study of dopamine presynaptic and postsynaptic elements. Other neurotransmitter abnormalities, however, have been observed in this and other basal ganglia diseases. This chapter will focus on the normal distribution of neurotransmitter receptors in human basal ganglia and the receptor abnormalities observed in Parkinson’s disease.
A general consensus exists as to the important role played by the basal ganglia in the control of voluntary movements. Dysfunction of the basal ganglia is responsible for a wide variety of movement disorders, e.g. Parkinson’s disease, chorea and dystonia. The basal ganglia exhibit a high level of structural complexity. Several neurotransmitter systems coexist in the basal ganglia and provide the substrate for signalling between the constituent nuclei. A characteristic of the chemical signalling within the basal ganglia is that peptides are often co-transmitted with amino acids or catecholamine. Within the basal ganglia, the striatum is a rich source of such peptidergic systems. However, very little is known regarding their functional significance in the control of movement or in the pathophysiology of movement disorders.
Parkinson's disease (PD) is the second most common age-related neurodegenerative disease resulting from the death of dopaminergic neurons in the substantia nigra pars compacta (SNc) which innervate all of the neuclei of the basal ganglia. Cell loss in the SNc causes a consequent striatal dopamine (DA) deficiency, leading to a cascade of functional changes in basal ganglia circuitry and ultimately the development of the cardinal features of PD, including resting tremor, bradykinesia, postural instability and rigidity, along with non-motor symptoms including cognitive, affective, psychiatric, and autonomic problems. DA replacement with the Levodopa (L-DOPA) is still a gold standard for symptomatic therapy of PD. However, the side effects of long-term use of L-DOPA are obvious. The emerging non- dopaminergic treatments for PD are becoming a scheduling program for the pipe- lines of drug development and clinical trials. The abundant expression of opioid receptors and their endogenous ligands, especially those for the δ-opioid system within the basal ganglia, has attracted much attention for its contribution to neurodegenerative diseases such as PD. In this article, we first discussed the relevant functional neuroanatomy and circuitry of the basal ganglia, and the evolving PD models of the basal ganglia. We then retrospected the studies on the location and expression of the δ-opioid system within the basal ganglia in health and PD, opioidinduced regulation of neurotransmitter release in basal ganglia, effects of pharmacological δ-opioid receptor (DOR) manipulation on PD and levodopa-induced dyskinesia (LID), and the effects of DOR activation on dopaminergic neuron injury/cellular model of PD. Based on these analyses, we believe that δ-opioid signaling is generally a beneficial, not detrimental factor for motor complications in PD. However, more in-depth investigations are expected to provide more solid and direct evidence for this benefit and to develop therapeutic strategies against PD by targeting δ-opioid system.
The role of enkephalin and the opioid receptors in modulating GABA release within the rat globus pallidus (GP) was investigated using whole-cell patch recordings made from visually identified neurons. Two major GP neuronal subtypes were classified on the basis of intrinsic membrane properties, action potential characteristics, the presence of the anomalous inward rectifier (I-h), and anode break depolarizations. The mu opioid receptor agonist [D-Ala(2)-N-Me-Phe(4)-Glycol(5)]enkephalin (DAMGO) (1 mu M) reduced GABA(A) receptor-mediated IPSCs evoked by stimulation within the striatum. DAMGO also increased paired-pulse facilitation, indicative of presynaptic mu opioid receptor modulation of striatopallidal input. In contrast, the delta opioid agonist D-Pen-[D-Pen(2,5)]-enkephalin (DPDPE) (1 mu M) was without effect. IPSCs evoked by stimulation within the GP were depressed by application of [methionine 5']-enkephalin (met-enkephalin) (30 mu M) Met-enkephalin also reduced the frequency, but not the amplitude, of miniature IPSCs (mIPSCs) and increased paired-pulse facilitation of evoked IPSCs, indicative of a presynaptic action. Both DAMGO and DPDPE reduced evoked IPSCs and the frequency, but not amplitude, of mIPSCs. However, spontaneous action potential-driven IPSCs were reduced in frequency by met-enkephalin and DAMGO, whereas DPDPE was without effect. Overall, these results indicate that presynaptic mu opioid receptors are located on striatopallidal terminals and pallidopallidal terminals of spontaneously firing GP neurons, whereas presynaptic delta opioid receptors are preferentially located on terminals of quiescent GP cells. Enkephalin, acting at both of these receptor subtypes, serves to reduce GABA release in the GP and may therefore act as an adaptive mechanism, maintaining the inhibitory function of the GP in basal ganglia circuitry.
It appears to be a general rule of pharmacology that the number of receptors for a hormone or neurotransmitter in a given tissue is not fixed, but may fluctuate in response to a change in the level of receptor activation, or a change in the requirement of the tissue for receptor activation. The reported examples of alterations in receptor number following experimental manipulation cover more or less the entire range of hormone and neurotransmitter systems, and opioid receptors have been found to be as subject to modulation as any other receptor type.
The development of biochemical techniques to quantitatively determine the interaction of drugs or neurotransmitters with their receptor site(s) was understandably met with enthusiasm by the scientific community. Homogenate binding studies provided a wealth of biochemical information, but only a limited amount of neuroanatomical data. The major problem was the limited resolution associated with macroscopic dissections, which resulted in only very general localizations of drug and neurotransmitter receptors. The development of autoradiography with its microscopic resolution was a major advance. Technical issues as well as the identification of various subtypes of opioid receptors have greatly complicated the interpretation of autoradiographic studies over the past decade. Initially, most investigators assumed the presence of a single class of receptor, as suggested by standard homogenate binding assays. To facilitate their studies, investigators chose radioligands upon the basis of their potency and technical factors, such as the ratio of specific to nonspecific binding, not aware that some compounds nonselectively labeled a variety of opioid receptor subtypes, whereas others were relatively selective. It soon became apparent that the various opioid receptor subtypes mediated different actions, underscoring the importance of establishing the differential distributions of subtypes of opiate binding sites.
This chapter describes the chemical composition of the basal ganglia. The basal ganglia comprise a distributed set of forebrain structures that are tightly interconnected with the cerebral cortex and thalamus, and with certain parts of the limbic system Most of the neurotransmitters, receptors, and regulatory molecules in the basal ganglia are differentially distributed with respect to the major functional subdivisions of the basal ganglia, including their input-output pathways, their macroscopic compartments, and their individual cell types. The basal ganglia and their allied nuclei together include many of the large subcortical structures of the forebrain. The largest of these is the striatum, itself made up of the caudate nucleus, the putamen, and a differentiated ventral part that includes the nucleus accumbens septi and the contiguous gray matter. The globus pallidus (pallidum) is the principal target of striatal outflow and forms a core structure of the basal ganglia.
The distribution of μ-opioid receptors, selectively labeled in vitro with a monoiodinated Met-enkephalin analog ([125I]FK 33-824), was analyzed by light and electron microscopic radioautography in sections from the neostriatum of the rat. In the light microscope, patches of high receptor densities were detected amidst a moderately labeled matrix. The number of silver grains, as counted in 1-μm thick plastic-embedded sections, was 3 times greater inside the patches than in the intervening matrix. In both compartments, the proportion of labeled binding sites associated with the neuropil was significantly higher (> 70%) than that associated with nerve cell bodies or myelinated fascicles. Quantitative analyses of electron microscopic radioautographs revealed that the majority of silver grains corresponding to specifically bound [125I]FK molecules originated from radioactive sources associated with apposed neuronal membranes. Of the total number of specific binding sites, 53% was associated with axodendritic, 18% with axoaxonic and 3% with axosomatic interfaces. The occurrence of multiple labeled foci along the plasma membrane of certain perikarya and dendrites suggested that some of the binding sites might be associated with somato/dendritic elements. The high incidence of labeling along axoaxonic interfaces indicated that others were linked to the membrane of axons and/or axon terminals. A major finding of the present study was that only a small proportion of specific FK binding sites (7% of total) was associated with synaptic junctions. Labeled synapses were primary of the asymmetric type and were found predominantly on dendritic branches and spines. A few were observed on nerve cell bodies. Labeled symmetric synapses were rare and encountered exclusively on dendritic branches. The high frequency with which specifically labeled binding sites were found to be associated with neuronal interfaces involving axonal processes strongly suggests that even if non-junctional these binding sites correspond to functional receptors. Whether these receptors are activated by endogenous ligand molecules released by the labeled terminals themselves or from terminals located at a distance from the labeled interfaces remains to be determined.
The present study aimed to characterize the ability of the novel delta opioid peptide (DOP) receptor agonist H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) to attenuate motor deficits in 6-hydroxydopamine (6-OHDA) hemilesioned rats. Lower doses (0.1-10 microg/kg) of UFP-512 administered systemically (i.p.) stimulated stepping activity in the drag test and overall gait abilities in the rotarod test whereas higher doses (100-1000 microg/kg) were ineffective or even worsened Parkinsonism. Microdialysis coupled to an akinesia test (bar test) was then used to determine the circuitry involved in the motor actions of UFP-512. An antiakinetic dose of UFP-512 (10 microg/kg) decreased GABA in globus pallidus (GP) as well as GABA and glutamate (GLU) release in substantia nigra reticulata (SNr). On the other hand, a pro-akinetic dose (1000 microg/kg) of UFP-512 increased pallidal GABA, simultaneously producing a decrease in GABA and an increase in nigral GLU release. Moreover, to test the hypothesis that changes in motor behavior were associated with changes in nigro-thalamic transmission, amino acid release in ventromedial thalamus (VMTh, a target of nigro-thalamic GABAergic projections) was also measured. The anti-akinetic dose of UFP-512 reduced GABA and increased thalamic GLU release while the pro-akinetic dose increased GABA without affecting thalamic GLU release. Finally, regional microinjections were performed to investigate the brain areas involved in motor actions of UFP-512. UFP-512 microinjections into GP increased akinesia whereas UFP-512 microinjections into SNr reduced akinesia. Furthermore, the selective DOP receptor antagonist naltrindole (NTD) increased akinesia when injected into either area, GP being more sensitive. We conclude that UFP-512, depending on dose, improves or worsens motor activity in hemiparkinsonian rats by acting differentially as a DOP receptor agonist in SNr and a DOP receptor antagonist in GP, ultimately decreasing or increasing the activity of nigro-thalamic GABAergc output neurons, respectively.
The delta opioid peptide (DOP) receptor has been proposed as a target in the symptomatic therapy of Parkinson's disease. However, the circuitry underlying the antiparkinsonian action of DOP receptor agonists and their site of action have never been adequately investigated. Systemic administration of the DOP receptor agonist (+)-4-[(alphaR)-alpha-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N-N-diethylbenzamide (SNC-80) attenuated akinesia/bradykinesia and improved motor activity in 6-hydroxydopamine hemilesioned rats. Opposite effects were produced by the selective DOP receptor antagonist naltrindole (NTD), suggesting that endogenous enkephalins tonically sustain movement under parkinsonian conditions. Microdialysis revealed that SNC-80 reduced GABA release in globus pallidus (GP) while NTD elevated it. Moreover, SNC-80 reduced GABA and glutamate release in substantia nigra reticulata (SNr) whereas NTD reduced GABA without affecting glutamate release. The bar test coupled to microdialysis showed that perfusion with NTD in SNr but not GP or striatum prevented the antiakinetic effect of systemic SNC-80 and its neurochemical correlates. Consistently, microinjections of SNC-80 into SNr or bicuculline in GP attenuated parkinsonian-like symptoms while SNC-80 microinjections in GP or striatum were ineffective. This study demonstrates that nigral DOP receptors mediate antiparkinsonian actions of SNC-80 and challenges the common view that DOP receptor agonists solely attenuate parkinsonism via pallidal mechanisms.
Bilateral microinjections of morphine hydrochloride (10; 20; 30 micrograms/0.5 microliter/side) or saline were aimed at three different regions of the rat globus pallidus: dorsal, medial, ventral. Before and at various intervals after intrapallidal morphine (15; 30; 60; 90; 180 min), estimation of pain threshold was made by the hot plate procedure. Dose-dependent morphine analgesia was elicited from all three regions injected. Differences between the pallidal areas as to the intensity and duration of the drug's effect were noticed. Pretreatment with subcutaneous naloxone (1 mg/kg, s.c.) inhibited the morphine (20 micrograms) analgesia elicited from the medial and dorsal pallidum; it decreased and delayed the effect of morphine injected into the ventral pallidum. The results suggest that the three pallidal areas tested are involved to a different degree (medial/dorsal greater than ventral) in the morphine analgesia mediated by opiate receptors.
The distribution of delta opioid receptors, selectively labelled in vitro with the photoaffinity probe monoiodo azido-DTLET ([D-Thr2,pN3Phe4, Leu5]enkephaly-Thr6), was analyzed by light and electron microscopic radioautography in sections from rat neostriatum. Preliminary experiments indicated that up to 65% of specific 125I-azido-DTLET binding to rat striatal sections was still detectable following prefixation of the brain with 0.5% glutaraldehyde. These experiments also showed that up to 20-30% of the specifically bound radioactivity was covalently linked following ultraviolet irradiation and was thereby retained in tissue during subsequent postfixation and dehydration steps. Accordingly, the topographic distribution of the covalently attached azido-DTLET molecules was similar to that seen in fresh frozen sections and characteristic of that previously described for delta sites. Light and electron microscopic examination of the label in prefixed, striatal sections irradiated with ultraviolet light revealed that a significant proportion of specifically bound 125I-azido-DTLET molecules was intraneuronal. Specifically, 16% of the labelled binding sites were found in dendrites, 12% in perikarya and 4% in axon terminals. These results suggest that an important proportion of delta opioid binding sites labelled in the neostriatum correspond to receptors that are undergoing synthesis, transport and/or recycling. They also imply that a major fraction of delta sites are associated with intrastriatal neurons, as opposed to afferent axons. Approximately 44% of the labelled binding sites were associated with neuronal plasma membranes. Although most of these were found at the level of axodendritic (20%) and dendrodendritic (7%) appositions, comparison of the labelling incidence of these two compartments with their frequency of occurrence in tissue suggested that delta sites are fairly widely dispersed along neuronal plasma membranes. Only a small proportion (smaller than that of mu or kappa sites labelled in the same region) was associated with synaptic specializations. These results support the concept that delta receptors correspond to molecular entities that are distinct from mu and kappa sites and suggest that delta ligands act primarily nonjunctionally on the plasma membrane of striatal neurons.
Endogenous opiates modulate activity in the mesocorticolimbic dopaminergic system, and this interaction is thought to underlie major aspects of motoric, reward-seeking, and stress-coping behaviors. We sought to determine the ultrastructural substrate for this modulatory action at the level of dopaminergic perikarya in the rat ventral tegmental area (VTA). Using a dual-labeling, immunoperoxidase and immunogold-silver method, we localized antisera directed against leu5-enkephalin (ENK) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) in acrolein-fixed sections through the VTA. ENK-like immunoreactivity (ENK-LI) was visualized within unmyelinated axons and in axon terminals. In terminals, ENK-LI was densely localized to one or more dense-cored vesicles and either densely or lightly detected surrounding small clear vesicles. Immunoreactive dense-cored vesicles were occasionally associated with the presynaptic specialization but were more frequently detected at distant sites along the plasmalemmal surface, often in apposition to astrocytic processes. ENK-immunoreactive terminals formed both symmetric and asymmetric synapses, most frequently on large proximal dendrites. Direct appositions without glial separation were also detected between terminals containing ENK-LI and other ENK-labeled or unlabeled terminals. In contrast to ENK-LI, immunolabeling for TH was primarily detected within perikarya and dendrites in the VTA. Of the ENK-immunoreactive terminals that formed synaptic contacts in single sections, approximately 50-60% were in association with TH-labeled dendrites. The remainder formed synapses on dendrites lacking detectable immunoreactivity for TH. Multiple ENK-immunoreactive terminals occasionally formed convergent synaptic contacts on single TH-labeled or unlabeled dendrites. Furthermore, individual ENK-labeled terminals sometimes formed divergent contacts on two TH-labeled or unlabeled dendrites. When a single ENK-immunoreactive terminal made dual synaptic contacts on TH-labeled dendrites, the latter were usually in close apposition to one another. These findings represent the first ultrastructural demonstration that opioid peptide-containing terminals provide a direct synaptic input to dopaminergic, as well as nondopaminergic, neurons in the VTA. In addition, the morphological evidence suggests that endogenous opioid peptides (1) may be released from nonsynaptic sites, (2) may modulate the release of transmitters from other terminals, and/or (3) may synchronize the activity of multiple neuronal targets in the VTA. These results provide a number of morphological substrates through which opiates may directly or indirectly regulate activity in mesocorticolimbic dopaminergic pathways.
Dopaminergic afferents to the dorsal striatum, caudate-putamen nuclei, are known to modulate the levels and synthesis of endogenous opiate peptides (Leu5 and Met5-enkephalins). We examined the dual immunocytochemical localization of antisera raised against Leu5-enkephalin and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the cellular substrates for these and/or other functional interactions. The antisera were identified by combined immunogold-silver and immunoperoxidase labeling in single coronal sections through the caudate-putamen nuclei of adult rats. These animals were given intraventricular injections of colchicine, and the brains were fixed by acrolein perfusion prior to immunocytochemical labeling. By light microscopy, perikarya and processes containing enkephalin-like immunoreactivity (ELI) were seen in close proximity to varicose processes immunoreactive for TH. Electron microscopy further demonstrated that the ELI was localized to perikarya, dendrites, and axon terminals, whereas the TH was exclusively in axons and terminals. The dendrites containing ELI were postsynaptic to terminals that were either (1) without detectable immunoreactivity, or (2) immunoreactive for TH or enkephalin. Nonsynaptic portions of the dendrites containing ELI were covered with astrocytic processes or were in direct apposition to unlabeled dendrites. Terminals containing ELI were densely immunoreactive and were in direct contact with (1) unlabeled and occasionally enkephalin-labeled proximal dendrites, and (2) TH-labeled and unlabeled terminals. In comparison with the opiate terminals, most catecholaminergic terminals were lightly immunoreactive for TH and usually contacted more distal unlabeled dendrites or spines and, more rarely, dendrites containing ELI. In a few favorable planes of section, the terminals containing ELI and those containing TH (1) converged on common unlabeled dendrites, or (2) formed dual contacts on two different labeled or unlabeled targets. Junctions formed by terminals containing ELI and TH were sometimes characterized by symmetric synaptic densities. However, numerous other dendritic and all axonal appositions were without recognized membrane densities. The findings of the study provide anatomical substrates for multilevel interactions between catecholamines, mostly dopamine, and enkephalin in rat dorsal striatum. These include (1) monosynaptic input from dopaminergic terminals to neurons containing enkephalin, (2) presynaptic modulation of transmitter release through axonal appositions, and (3) dual regulation of common targets through convergent input. In addition, the findings suggest that both enkephalin and dopamine may have similar modulatory roles in synchronizing the activity of dual targets postsynaptic to individual axon terminals. Alterations in any one of these multiple types of interactions could account for noted motor or sensory symptoms in neurological disorders characterized by depletion of dopamine or endogenous opiate peptides, or both.
In vivo electrophysiological recording techniques were employed to examine responses of ventral pallidum/substantia innominata (VP/SI) neurons to systemic and local administration of morphine. Using a cumulative dosing protocol, intravenous administration (0.1-30 mg/kg i.v.) produced a suppression of firing in 82% of neurons tested. The suppression was dose-related and blocked by the opioid antagonist, naloxone. In contrast, microiontophoretic applications of morphine resulted in current-related suppression (32% of neurons tested) or excitation (26%). Concurrent application of naloxone attenuated or blocked both effects of local morphine application. It was demonstrated that acute tolerance did not develop with repeated morphine exposures following either systemic or local administration. The present findings establish the sensitivity of VP/SI neurons to morphine and provide functional relevance at the level of a single neuron for opioid peptides and their receptors in this region. As reported for most other opioid-receptive brain areas, neuronal rate suppression was the predominate response observed, and it is proposed that excitations to iontophoresed morphine reflect a disinhibitory phenomenon. The differential morphine-induced rate changes, and number of responding neurons, observed with systemic vs. iontophoretic morphine administration suggest that extra-VP/SI regions that also are opioid sensitive can subsequently direct neuronal responsiveness to opioids within the VP/SI.
Quantitative autoradiography was used to study [3H]naloxone binding in the striatum of normal monkeys and monkeys made hemiparkinsonian by the unilateral infusion of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The density of [3H]naloxone binding sites was significantly higher in the caudate and putamen on the MPTP-treated side of hemiparkinsonian monkeys, as compared with binding on the untreated side and in the striatum of normal monkeys. A more extensive patchy distribution of binding sites was evident throughout the striatum on the MPTP-treated side than seen in the striatum of the untreated side or in normal striatum.
In conclusion, GABAA receptors containing the alpha 1 subunit are localized on postsynaptic neurons in the ventral pallidum, mainly in the dorsolateral compartment and on presynaptic terminals in the nucleus accumbens. mu-opioid receptors are localized on postsynaptic neurons in both the nucleus accumbens and ventral pallidum, and therefore may be regulating presynaptic release of enkephalin from the accumbens-pallidal projection. Discrete lesions in the dorsomedial core of the nucleus accumbens will upregulate GABAA receptors in the dorsolateral compartment of the nucleus accumbens in a fashion similar to the upregulation of GABAA receptors in the globus pallidus after striatal lesions. However, larger lesions of the lateral core projection to the dorsolateral compartment of the ventral pallidum do not upregulate the GABAA receptors, suggesting that the mechanisms for upregulation of GABAA receptors are specific to the dorsomedial core or a smaller lesion. The uniqueness of the compartments within the nucleus accumbens and the ventral pallidum are supported by these receptor and mRNA analyses.
The present study demonstrates that desacetyllevonantradol, a synthetic cannabinoid analog, reduces cyclic AMP levels in rat striatal slices stimulated with vasoactive intestinal peptide or SKF 38393, a D1-dopamine agonist. Desacetyllevonantradol and the D2 agonist LY 171555 both inhibited D1-stimulated cyclic AMP accumulation in the striatum. Spiperone, a specific D2-dopamine antagonist, fully reversed the inhibitory effect of LY 171555 but not that of desacetyllevonantradol, indicating that this cannabinoid response is not occurring through a D2-dopaminergic mechanism. Morphine also inhibited cyclic AMP accumulation in striatal slices stimulated with either SKF 38393 or vasoactive intestinal peptide. Naloxone, an opioid antagonist, fully reversed the effect of morphine but not that of desacetyllevonantradol, indicating that cannabinoid drugs are not acting via a mechanism involving opioid receptors. The response to maximally effective concentrations of desacetyllevonantradol was not additive to that of maximally effective concentrations of either morphine or LY 171555, suggesting that dopaminergic, opioid, and cannabinoid receptors may be present on the same populations of cells.
A number of different neuroactive substances have been found in striatal projection neurons and in fibers and terminals in their target areas, including substance P (SP), enkephalin (ENK), and dynorphin (DYN). In a preliminary report on birds and reptiles, we have suggested that SP and DYN are to a large extent found in the same striatal projection neurons and that ENK is found in a separate population of striatal projection neurons. In the present study, we have examined this issue in more detail in pigeons and turtles. Further, we have also explored this issue in rats to determine whether this is a phylogenetically conserved feature of basal ganglia organization.
Simultaneous immunofluorescence double‐labeling procedures were employed to explore the colocalization of SP and DYN, SP and ENK, and ENK and DYN in striatal neurons and in striatal, nigral, and pallidal fibers in pigeons, turtles, and rats. To guard against possible cross‐reactivity of DYN and ENK antisera with each others' antigens, separate double‐label studies were carried out with several different antisera that were specific for DYN peptides (e.g., dynorphin A 1–17, dynorphin B, leumorphin) or ENK peptides (leucine‐enkephalin, metenkephalin‐arg ⁶ ‐gly ⁷ ‐leu ⁸ , methionine‐enkephalin‐arg ⁶ ‐phe ⁷ ). The results showed that SP and DYN co‐occur extensively in specific populations of striatal projection neurons, whereas ENK typically is present in different populations of striatal projection neurons. In pigeons, 95‐99% of all striatal neurons containing DYN were found to contain SP and vice versa. In contrast, only 1‐3% of the SP+ striatal neurons and no DYN neurons contained ENK. Similarly, in turtles, greater than 75% of the SP+ neurons were DYN+ and vice versa, whereas ENK was observed in fewer than 5% of the SP+ neurons and 2% of the DYN+ neurons. Finally, in rats, more than 70% of the SP+ neurons contained DYN and vice versa, but ENK was found in only 5% of the SP+ neurons and in none of the DYN+ perikarya. Fiber double‐labeling in the striatum and its target areas (the pallidum and substantia nigra) was also consonant with these observations in pigeons, turtles, and rats.
These results, in conjunction with studies in cats by M.‐J. Besson, A. M. Graybiel, and B. Quinn (1986; Soc Neurosci. Abs. 12 :876) strongly indicate that the co‐occurrence of SP and DYN in large numbers of striatonigral and striatopallidal projection neurons is a phylogenetically widespread, and therefore evolutionarily conserved, feature of basal ganglia organization. These populations of SP/DYN‐containing projection neurons appear to be distinct from those containing ENK. The evolutionarily conserved nature of such widespread SP/DYN co‐occurrence in striatal neurons that contain either of these peptides suggests that such co‐occurrence is an important functional characteristic of the basal ganglia. The precise significance of such co‐occurrence in terms of neurotransmission between these neurons and striatal target neurons, however, requires further study.
Two largely separate populations of neuropeptide-containing striatonigral projection neurons have been distinguished in pigeons, one population whose neurons contain substance P (SP) and dynorphin (DYN) and a second population whose neurons contain enkephalin (ENK) (Reiner, '86a; Anderson and Reiner, 90a). In the present study, we investigated the abundance of these two types of neurons relative to all striatonigral projection neurons by combining retrograde labeling by the fluorescent dye fluorogold with immunofluorescence labeling for SP and ENK.
Pigeons received large intranigral injections of fluorogold to retrogradely label the striatonigral projection neurons, and several days later they were treated with colchicine (32 hours before transcardial perfusion). Adjacent series of sections through the basal ganglia were labeled for SP and ENK using immunofluorescence techniques. The tissue was examined using fluorescence microscopy and the percentages of retrogradely labeled neurons containing either SP or ENK were quantified. We found that 85–95% of the fluorogold-labeled striatonigral neurons were SP+, whereas only 1–4% were ENK+.
Thus the majority of striatonigral projection neurons in pigeons appear to contain SP, whereas a small percentage contain ENK. Only a small percentage of striatonigral neurons did not contain either. Since striatal projection neurons also contain GABA (Reiner, '86b), the present results suggest that a high percentage of striatonigral projection neurons coexpress SP, DYN and GABA, whereas a small fraction coexpress ENK and GABA. The available data are consistent with the conclusion that this is true in reptilian and mammalian species as well.
The present study investigated the effects of a striatal lesion induced by kainic acid on the striatal modulation of dopamine (DA) release by mu- and delta-opioid peptides. The effects of [D-Pen2,D-Pen5]-enkephalin (DPDPE) and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAGO), two highly selective delta- and mu-opioid agonists, respectively, were studied by microdialysis in anesthetized rats. In control animals both opioid peptides, administered locally, significantly increased extracellular DA levels. The effects of DPDPE were also observed in animals whose striatum had been previously lesioned with kainic acid. In contrast to the effects of the delta agonist, the significant increase induced by DAGO was no longer observed in lesioned animals. These results suggest that delta-opioid receptors modulating the striatal DA release, in contrast to mu receptors, are not located on neurons that may be lesioned by kainic acid.
The neurotransmitter organization of striatal projection neurons appears to be less complex than once thought. Only 4 major evolutionarily conserved populations appear to be present. The neurons of two of these populations contain SP, DYN and GABA, with one of these two populations consisting of striatonigral projection neurons and the other of striatopallidal projection neurons. The two additional major populations of striatal projection neurons consist of striatopallidal and striato-nigral neurons that both contain both ENK and GABA. Although these conclusions greatly simplify the understanding of the organization of striatal projection neurons by suggesting that only a few major populations are present, these conclusions complicate understanding of neurotransmission between these neurons and their target areas by suggesting that each neuron utilizes multiple neuroactive substances to influence target neurons. Further studies will therefore be required to explore the mechanisms of neurotransmission by which striatal neurons communicate with their target areas.
Discrete quinolinic acid lesions in the nucleus accumbens altered [3H]muscimol binding to gamma-aminobutyric acid receptors, [125I]neurotensin binding to neurotensin receptors, [125I]Tyr-D-Ala-Gly-NMePHe-Gly-OH binding to mu-opioid receptors, and [3H]quinuclidinyl benzilate binding to muscarinic receptors. Within lesions of the lateral accumbens core, [3H]muscimol binding increased and [125I]Tyr-D-Ala-Gly-NMePhe-Gly-OH, [125I]neurotensin and [3H]quinuclidinyl benzilate binding decreased. Lesions of the medial nucleus accumbens resulted in decreased [125I]Tyr-D-Ala-Gly-NMePhe-Gly-OH and [3H]quinuclidinyl benzilate binding while no alterations were observed for [3H]muscimol or [125I]neurotensin binding. These data support anatomical distinctions between medial and lateral nucleus accumbens. Destruction of intrinsic neurons in the dorsomedial nucleus accumbens core increased [3H]muscimol binding in the dorsal rim of the ventral pallidum and the rostral globus pallidus without altering [125I]Tyr-D-Ala-Gly-NMePhe-Gly-OH binding. Destruction of neurons in the lateral nucleus accumbens core or medial shell did not alter [3H]muscimol binding in the ventral pallidum. The lack of upregulation in gamma-aminobutyric acid receptors suggests that the gamma-aminobutyric acid-containing projection from the dorsomedial core to the dorsal rim of the ventral pallidum differs from the projection from the lateral accumbens core and medial shell to the more ventral regions of the pallidum. Fluoro-gold retrograde tracer histochemistry confirmed the specific projection from the dorsomedial core to the dorsal ventral pallidum; and from the shell of the nucleus accumbens to more ventral regions of the ventral pallidum.
The plasticity of the beta 1- and beta 2-adrenergic receptor subtypes was examined in the interpeduncular nucleus (IPN) of the adult rat. The beta-adrenergic receptor antagonist 125I-pindolol (125I-PIN) was used in conjunction with the selective subtype antagonists ICI 118,551 and ICI 89,406 to determine the subnuclear distribution of beta 1- and beta 2-adrenergic receptors in this nucleus and to correlate the receptor distribution with the distribution of both noradrenergic afferents from the locus coeruleus (LC) and non-noradrenergic afferents from the fasiculus retroflexus (FR). The density of these binding sites was examined following lesions that decreased (LC lesions) or increased (FR lesions) the density of the noradrenergic projection in the IPN. Quantitative radioautography indicated that beta 1-labeled binding sites account for the larger percentage of binding sites in the IPN. The beta 1-binding sites are densest in the those subnuclei that receive a noradrenergic projection from the LC: the central, rostral, and intermediate subnuclei. beta 1-binding sites are algo homogeneously distributed throughout the lateral subnuclei, where there is no detectable noradrenergic innervation. beta 2-binding sites have a more restricted distribution. They are concentrated in the ventral half of the lateral subnuclei, where they account for 70% of total 125I-PIN binding sites. beta 2-binding sites are also present along the ventral border of the IPN. Some of this labeling extends into the central and intermediate subnuclei. Bilateral lesions of the LC, which selectively remove noradrenergic innervation to the IPN, result in an increase in the beta 1-binding sites. Bilateral lesions of the FR, which remove the major cholinergic and peptidergic input from the IPN, elicit an increase in noradrenergic projections and a decrease in beta 1-binding sites. beta 1-binding sites thus exhibit both up-regulation and down-regulation which is correlated with the density of the noradrenergic projection. Our results suggest, therefore, that the density of beta 1-binding sites is regulated by noradrenergic input. beta 2-binding sites increase in density in response to both the LC and FR lesions, suggesting that they are postsynaptic to both of these afferents. The distribution suggests that some of these binding sites may reflect binding to glial cells. The beta 2-binding sites may therefore be regulated by both noradrenergic and non-noradrenergic mechanisms.
The effects of the microiontophoretic application of dynorphin A-(1-13) (DYN 13) and the benzomorphans ethylketocyclazocine (EKC), bremazocine and MRZ 2549, (kappa) opioid agonists, and of morphine and morphiceptin, (mu) opioid agonists, were compared on spontaneous or glutamate-evoked discharge of globus pallidus (GP) neurons in rat. Our results demonstrate that mu and kappa opioid agonists are able to depress the excitability of pallidal neurons, possibly by interacting with mu and kappa opioid receptor subtypes, respectively. In addition, the mu agonists and dynorphin A-(1-13), but not the benzomorphans, enhanced the excitability of a number of pallidal neurons. We have proposed a presynaptic site as the basis for this opioid-induced excitation, possibly also mediated by a mu opioid receptor. The selectivity of dynorphin A-(1-13) for benzomorphan kappa opioid receptors in the rat GP appears to be low and dynorphin A-(1-13) may elicit effects that are different from those produced by the benzomorphan kappa agonists by virtue of its ability to interact with other opioid receptor subtypes, for example mu opioid receptors.
Opiate receptors have been localized autoradiographically to many regions of the rat central nervous system. The interpeduncular nucleus has an especially high concentration of these receptors. We used [3H]naloxone and [125I] [D-Ala2,MePhe4,Glyol5]enkephalin as ligands to map the distribution of opiate receptors among the subnuclei of the interpeduncular nucleus. The rostral subnucleus contains label that is densest dorsally. More caudally, high densities of opiate binding sites are found in the lateral, rostral, and central subnuclei. The dorsal subnucleus contains a moderate density of binding sites and the intermediate subnuclei contain almost none. Opiate receptors have also been localized to the medial habenulae and the fasciculi retroflexi, which provide a major afferent input to the interpeduncular nucleus. Lesions of the fasciculi retroflexi decreased the density of opiate binding sites in the rostral and lateral subnuclei of the interpeduncular nucleus. There were no changes seen in the dorsal, intermediate or central subnuclei. These results suggest that a minority of opiate receptors in the interpeduncular nucleus are located presynaptically on fasciculi retroflexi axons. Immunocytochemical studies have demonstrated that the rat interpeduncular nucleus contains substance P, serotonin and enkephalin, and each has a distinct subnuclear distribution. Although the opiate binding sites have a wider distribution than substance P, serotonin, or enkephalin individually, the pattern of opiate binding most closely parallels substance P distribution. The combined distribution of substance P, serotonin, and enkephalin is equivalent to that of the opiate binding sites.
The effects of mu- and delta-preferring agonists on adenylate cyclase activity have been investigated in vitro in homogenates of guinea pig cochleas. Morphine, Leu-enkephalin, D-Ala2, N-methyl-Phe4, Gly-ol5-enkephalin (DAGO) and D-Ser2-Leu-enkephalin-Thr (DSLET) each inhibited the synthesis of cyclic AMP. This effect was reversed by naloxone which had a greater affinity in blocking the effect of the mu-preferring agonists (morphine, DAGO) than in blocking the effect of the delta-preferring agonists (Leu-enkephalin, DSLET). Finally, no additive effects were observed when various combinations of two agonists were used. These results indicate that opioid receptors exist in the guinea pig cochlea and that they are negatively linked to adenylate cyclase. The different affinities shown by naloxone to reverse the inhibition induced by the mu- and delta-preferring agonists suggest that morphine and DAGO act through mu-receptors, whereas Leu-enkephalin and DSLET act through delta-receptors. Since no additive effects have been found when combining two different agonists, it can be hypothesized that the mu- and delta-receptors are coupled to the same pool of adenylate cyclase. It may be proposed from these findings that in vivo enkephalins inhibit the synthesis of cyclic AMP via mu- and delta-receptors. However, whether this effect occurs at a presynaptic level (within opioid-containing olivocochlear varicosities) or at the postsynaptic level (within dendrites of the primary auditory neurons) remains to be determined.
The cellular localization of the rat brain neutral endopeptidase (NEP, EC 3.4.24.11) was investigated by quantitative autoradiography of the enzyme inhibitor ([3H]HACBO-Gly) after lesions of the striatum, nigrostriatal and corticostriatal pathways. The effect of these lesions on NEP levels was compared with that on δ and μ opioid receptors, selectively labeled with [3H]Tyr-d-Thr-Gly-Leu-Thr ([3H]DTLET) and [3H]Tyr-d-Ala-Gly-MePhe-Glycinol ([3H]DAGO), respectively. Twenty-one days after injection of kainate in the caudate putamen (CP), the NEP level was locally decreased (52%) but the time course of this decrease was different from that of μ and δ opioid receptors: [3H]DAGO binding was diminished by 40% from day 2 whereas that of [3H]DTLET was reduced by 51% from day 7. Kainic acid injection in the CP induced in the globus pallidus (GP) and substantia nigra (SN) a distant reduction of the 3 opioid markers. Likewise after injection of colchicine in the CP, [3H]HACBO-Gly binding was decreased in the GP (60%) and SN (58%), [3H]DTLET binding was reduced by 54 and 55% in the GP and SN, respectively and [3H]DAGO labeling was diminished by 49% in the GP, and 58% in the SN. Finally, lesion of the nigrostriatal dopamine pathway by 6-hydroxydopamine did not induce any change of NEP level in the CP and GP whereas δ and μ opioid receptor levels were diminished respectively by 25 and 29% in the CP, and 45 and 39% in the GP, a new fiding of the present study. Taken together these data suggest that NEP is in part associated with striatal intrinsic neurons. In the GP and SN, a large part of NEP seems to be presynaptically associated with nerve terminals endowed with μ and δ opioid receptors, which originate from efferent striatal neurons. In contrast to opioid receptors in the CP, the NEP appears not to be associated with dopaminergic nerve terminals originating from the SN. Cortical ablation did not affect any of the opioid markers.
Recent studies have demonstrated that opioid receptors may be functional at early stages of ontogeny, and may modulate specific developmental functions. It is presently unknown, however, which particular opioid receptor subtype(s) may be involved. In the pre-ent study, we have used selective radioligand binding conditions in combination with quantitative autoradiography to examine the ontogeny of mu-, kappa- and delta-opioid receptors in the developing rat brain. Membrane binding data indicate that the affinities of mu-, kappa- and delta-sites for radiolabeled drugs are similar in neonatal and adult rats. mu- And kappa-receptors are present in significant densities during early neonatal periods, while delta-receptors appear much later. Autoradiographic data indicate that mu- and kappa-receptors appear early in the development of several brain regions, including the neostriatum, olfactory tubercle and rostral midbrain, and later in other regions such as the thalamus and hypothalamus. Whereas the densities of kappa-binding sites remain relatively constant throughout development, there is a transient appearance and/or redistribution of mu-receptors in several brain areas. delta-Receptors are present in low densities in the basal forebrain at birth. The level of delta-receptor binding increases markedly during the third postnatal week in all brain areas examined. The early appearance of mu- and kappa-receptors during the ontogeny of the brain suggests that these receptors, at least in part, mediate the developmental actions of exogenous and endogenous opioids.
Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. Mu1 binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and mu2 binding assays supported these observations. No change in delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.
The axonal transport of mu and delta receptors in the rat was examined by autoradiography in three fiber systems: vagus nerve, fasciculus retroflexus and corpus callosum. Following ligature or knife cut, sections were incubated with [125I]D-Ala2-MePhe4-Met(O)5-ol-enkephalin to label mu receptors or [125I]D-Ala2-D-Leu5-enkephalin in the presence of 30 nM oxymorphone to selectively label delta receptors. Ligature of the vagus and knife cut of the fasciculus retroflexus produced a time-dependent proximal build-up of mu receptors indicating anterograde axonal flow. In contrast, the corpus callosum has delta but not mu receptors, and these could not be demonstrated to undergo axonal flow. The results suggest that in the fiber tracts examined, mu but not delta opiate receptor subtypes are anterogradely transported toward nerve terminals where they may be inserted in presynaptic membranes.
This study was conducted in order to compare the effects of microiontophoretically-applied morphine and met-enkephalin (met-ENK) on spontaneous and/or glutamate-evoked activity of single globus pallidus (GP) neurons in locally anesthetized, paralyzed rats. The predominant effect of both morphine and met-ENK was a depression of pallidal neuronal activity. While very few GP neurons were excited by morphine (2/89), a small population of neurons was excited by met-ENK (16/89). Both the inhibitory and excitatory responses produced by morphine and met-ENK could be attenuated by the microiontophoretic application of naloxone. It was also found that morphine and met-ENK did not affect all GP neurons in a similar manner. When applied to the same neurons, morphine elicited depression in 11 of 16 GP neurons which were excited by the application of met-ENK. In contrast, neither of two GP neurons excited by morphine in this study displayed inhibition upon application of met-ENK. Thus the microiontophoresis of morphine and met-ENK to single GP neurons has demonstrated that these two substances can produce opposite effects when applied to the same neurons and suggests that two functionally distinct types of opiate receptor may exist within rat GP, one which mediates the inhibitory effects of morphine and met-ENK, possibly the classical mu (mu) receptor, and one that is preferentially selective to met-ENK and which mediates the excitatory effects of opiates within this region, possibly the classical delta (delta) receptor.
Chronic administration of iminodipropionitrile (IDPN) causes a persistent behavioral syndrome which consists of hyperactivity, vertical neck dyskinesias and lateral head twitches. D-Ala-D-Leu-enkephalin binding revealed a 26% decrease in the number but no change in the affinity of delta-opiate receptors in the striata of IDPN-treated rats. These findings are similar to those seen in the brains of patients with Huntington's disease. Further studies are needed to clarify the relationship of these findings to the phenomenology of the IDPN-induced dyskinetic abnormalities.
The use of very low concentrations of 125I-[D-Ala2,D-Leu5]enkephalin, [3H]naxolone, and [3H]dihydromorphine under similar conditions enables the measurement of different opiate binding sites in a rat brain membrane preparation. Data may suggest that the hydrophobic group of the phenylalanine residue of enkephalin could be responsible for the major structural difference between enkephalin and morphine for receptor recognition. This may also explain why N-cyclopropylmethylnoretorphine, which contains a hydrophobic group at the C-19 position of oripavine, binds to the enkephalin receptor better than naxolone and morphine.
In the mammalian central nervous system, high concentrations of Leu-enkephalin, an opioid pentapeptide, have been found within the globus pallidus and caudate-putamen, where opiate receptors also are found in abundance. Opiate analgesics and opioid peptides have been proposed to interact with known neurotransmitters in the neostriatum and globus pallidus. With the use of the peroxidase-antiperoxidase method, we examined the light microscopic and ultrastructural localiza- tion of immunoreactive Leu-enkephalin-containing neurons and axon terminals in the monkey caudate-putamen, globus pallidus, and substantia nigra. Only neostriatal neurons contained immu- noreactive Leu-enkephalin. Labeled somata were medium size (12 to 20 pm) and were located primarily in the caudate nucleus and the ventral and medial portions of the putamen. Immunoreac- tive Leu-enkephalin fibers were present, in order of greatest density, in the outer segment of the globus pallidus, substantia nigra pars reticulata, and neostriatum. Fiber staining in the inner pallidal segment was sparse. At the electron microscopic level, labeled neostriatal somata had relatively large, slightly indented nuclei and scant cytoplasm. Primary dendrites were smooth and distal branches had numerous spines. Together, these features have been shown previously in the monkey by the Golgi-EM technique to distinguish spiny type 1 neurons. In the neostriatum and outer segment of the globus pallidus, small diameter unmyelinated (about 0.2 pm) and myelinated (about 1 pm) axons contained immunoreactive Leu-enkephalin. Labeled boutons were 0.5 to 1.5 pm in size and had small clear round and/or ovoid vesicles and also large granular vesicles. The majority of labeled terminals appeared to make symmetric contacts. In the neostriatum, they formed synapses with unlabeled somata which had cytologic features of spiny type 1 and aspiny type 1 neurons and with the primary and distal branches of unlabeled spiny (shafts) and aspiny dendrites. Few immunoreactive boutons synapsed with unlabeled dendritic spines. Positive boutons were found also to synapse upon the axon hillocks and axon initial segments of unlabeled somata. Other types of axoaxonic synapses were not found. Some immunoreactive Leu-enkephalin terminals formed syn- apses with cell bodies and dendrites which also were positively labeled for Leu-enkephalin. Within the globus pallidus, nearly one-half of all axon terminals that ensheath and synapse with pallidal dendrites contained immunoreactive Leu-enkephalin. From the anatomic observations, it appears likely that immunoreactive Leu-enkephalin is found within medium size spiny neurons (spiny type 1 in the monkey), which are known to have axon collaterals intrinsic to the caudate-putamen and to project to the globus pallidus and substantia nigra. The high density of enkephalin-positive terminals found in the latter two structures is consistent with the presence of a striatofugal enkephalin pathway which may have a direct influence on the efferent systems originating in the globus pallidus
Slide-mounted sections of unfixed frozen rat brain can be labeled in vitro with [3H]naloxone to show the mu-like ligand selectivity characterized in previous studies. We have developed an autoradiographic technique using hot paraformaldehyde vapors to prevent diffusion of ligands with reversible binding. Resolution at the light level is sufficient to detect concordance between receptor patterns and terminal fields of axonal projections marked by tract-tracing techniques. The opiate receptor distribution suggests the existence of widespread intrinsic and several longer multisynaptic "opiatergic pathways within sensory and limbic circuits. One multisynaptic pathway may link olfactory structures with limbic circuits in the amygdala and habenula. Another may lie in limbic cortical structures. Opiate receptors are numerous also in sensory systems, and within primary sensory nuclei (visual, auditory, olfactory, somatic) they are found superficially in laminated structures. Together, the opiate receptors are well placed to control incoming sensory and subsequent limbic information processing.
We have observed two discrete populations of opiate receptors that are differently localized in rat brain. Morphine-like (mu) receptors, labeled by 125I-labeled [D-Ala-2MePhe4Met(O)5-ol]enkephalin, are concentrated selectively in lamina IV of the cerebral cortex, certain thalamic nuclei, and the periaqueductal grey, while delta receptors, labeled by 125I-labeled [D-Ala2-D-Leu5]enkephalin, are more diffused, having high densities in cerebral cortex, corpus striatum, amygdala, and olfactory tubercle. Because of similarities in their localizations, we propose that mu and delta receptors are respectively the physiologic receptors for [Met]- and [Leu]enkephalin neurons. These distributions reflect the different physiological functions attributed to mu and delta receptors and thus represent discrete functions of [Met]- and [Leu]enkephalin neurons.
A general technique is described for using slide-mounted unfixed tissue sections to characterize and visualize drug and neurotransmitter receptors in brain or other tissues. The preparation of material, from fresh frozen, unfixed brain to dried sections securely attached to slides, is described in detail. The tissue can be kept intact during incubation at varying temperatures in solutions containing radiolabeled ligand, ions, buffers, and allosteric effectors. Strategies are described for determining optimal stereospecific binding with highest signal-to-noise ratios and for determining that a "meaningful" receptor is being studied. Dry formaldehyde fixation by vapors from heated paraformaldehyde preserves the tissue quality and traps the ligand near its site on the receptor, permitting subsequent histological processing through alcohols, solvents, and aqueous media, including liquid nuclear track emulsion. Visualization of [3H]naloxone- or [3H]enkephalin-labeled opiate receptor distributions in rat and human brains is achieved by tritium-sensitive film or by classical "wet" emulsion autoradiography. The advantages of the film include its ease of use and the ability to quantify receptor density by densitometry which can be computer-assisted. The advantage of the emulsion is the greater resolution and the concomitant appearance of morphology in cell-stained sections. Examples of correlations of opiate receptor distributions which underlying cytoarchitecture illustrate the potential for receptor localization studies.
A quantitative autoradiographic technique for measuring the binding of [3H]muscimol to central nervous system GABA receptors is described using tritium-sensitive film. [3H]Muscimol binding was studied in primary and secondary striatal projection areas of rat brain following kainic acid lesions of the striatum. Seven days after the lesion, binding affinities in the striatum and its projection areas were not altered significantly. There was a loss of [3H]muscimol receptors in the striatum. Receptors increased in numbers in the ipsilateral globus pallidus (19%), entopeduncular nucleus (22%), and substantia nigra pars reticulata (38%). [3H]Muscimol binding was decreased in the ipsilateral anteroventrolateral and ventromedial (8%) thalamic nuclei. [3H]Muscimol binding in other brain areas (layer IV of the cerebral cortex, central gray, superior colliculus, and stratum moleculare of hippocampus) was not affected. The findings suggest that a loss of striatal innervation resulted in increased numbers of GABA receptors in striatal projection sites. It is further suggested that loss of inhibitory striatal inputs to neurons in the entopeduncular nucleus and substantia nigra pars reticulata may activate GABAergic projections to thalamus and thus result in decreased numbers of thalamic GABA receptors.
The synthetic peptide NH2-Tyr-Pro-Phe-Pro-CONH2 (morphiceptin), which is the amide of a fragment of the milk protein beta -casein, has morphinelike activities and is highly specific for morphine (mu ) receptors but not for enkephalin (delta ) receptors. It is as active as morphine in the guinea pig ileum but much less active in the mouse and rat vas deferens. The discovery of this specific morphine receptor ligand substantiates the hypothesis of multiple opiate receptors. The ligand, which may be of physiological significance since a very similar, or identical, activity can be detected in enzymatic digests of beta -casein, may prove useful for further investigation of the functions of opiate receptor subtypes.
Destruction of the rat Striatonigral GABA pathway by striatal kainic acid (KA) lesion resulted in both increased behavioural response to intranigral muscimol and elevated nigral 3H-GABA binding. Behavioural responses were correlated with both depletions of GABA and elevations in 3H-GABA binding. Elevated 3H-GABA binding was characterised by an increase in Bmax with no change in Kd for the high affinity component. Following the proposal of the striatal KA lesion as an animal model for Huntington's disease (HD),33H-GABA binding was studied in the nigra of post-mortem brains from 5 control and 4 HD patients. Bmax for the high affinitiy component was elevated by 136% (p<0.01) with no change in Kd. Low affinity binding parameters were unaltered. These results are consistent with the development of denervation supersensitivity of nigral GABA receptors in HD and its animal model. They demonstrate the adaptive capacity of the human GABA system and have implications for the therapeutic potential of GABA agonists in HD.
Opiate binding sites of the κ-subtype were visualized in guinea-pig brain sections in vitro autoradiography. κ-Binding sites, defined as [3H](−)-bremazocine binding in the presence of high concentrations of [D-Ala2, MePhe4, Gly-ol5]enkephalin and [D-Ala2, D-Leu5]enkephalin, were found in the cortical laminae V and VI, hippocampal dentate gyrus, and lateral habenulae. The distribution of κ-sites in the guinea-pig differs considerably from the distribution of μ- and δ-sites which others have found in the rat.
The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretretated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nuclear tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephelon, mensencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.
This report presents immunohistochemical evidence for a modular arrangement of enkephalin and substance P in the striatum of cats and kittens. In cross-sections from the adults, met-enkephalin immunoreactivity in the caudate nucleus was concentrated in discrete, variably shaped macroscopic patches (0.2–0.5 mm wide) spaced at roughly 0.7–1.0 mm intervals. Patches appeared only occasionally in the putamen but there were larger districts of high immunoreactivity in the nucleus accumbens. In sections incubated with antiserum to substance P, both patches of dense immunoreactivity and zones of low immunoreactivity were present in the caudate nucleus. Staining in the putamen was fairly homogeneous, while in the nucleus accumbens it was patterned and especially intense dorsally.A detailed comparison was made between the distribution of the enkephalin-rich patches and the circumscript zones of low acetylcholinesterase activity (striosomes) visible in serially-adjoining sections by the thiocholine method. There was a striking registration of the two sets of figures, nearly every cholinesterase-poor patch corresponding to an enkephalin-positive zone. Correspondences were more complicated in the substance P stain but many pale zones and some dark zones were aligned with the enkephalin-rich cholinesterase-poor figures. No comparable clumping of reaction product was visible in sections processed for somatostatin immunoreactivity.In the caudate nucleus of young kittens, small dark patches of enkephalin and substance P immunoreactivity also appeared. In the cholinesterase stain there were both light and dark patches, and it was mainly with the dark zones that the enkephalin-positive patches (and some substance P patches) were in register.We conclude that the caudate nucleus is divided up into pharmacologically distinct compartments, and that although these are present at birth, their final histochemical composition is established postnatally.
Knife cuts in the frontal plane separating the anterior part of the caudate-putamen from the globus pallidus resulted in marked decreases in substances P levels in the reticular part of the substantia nigra. More caudal knife cuts were required in order to effect maximal decreases in nigral glutamic acid decarboxylase levels. Thus, there is a clear anatomical dissociation between the striatal neurons which project to the reticular part of the substantia nigra and which contain SP, and the more caudally located GAD-containing striatal and pallidal neurons, all of which travel through the globus pallidus on their way to the substantia nigra.
An antiserum specific for the C-terminal region of dynorphin1–17 (DYN) was used to examine the distribution of this endogenous opioid peptide in the rat brain with the indirect immunofluorescence technique. DYN-positive nerve cell bodies and fibers were found in many nuclei in the spinal cord, medulla, mesencephalon, hypothalamus and forebrain. These findings indicate that a widespread system of DYN neurons is present in the brain distinct from the previously described enkephalin and endorphin systems.
The projections of the head of the striatum of the rat were studied by autoradiography and horseradish peroxidase (HRP) histochemistry. After injections of [³H]-leucine into the head of the striatum, accumulations of autoradiographic grains were observed over the neuropil of the globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra, pars reticulata (SNR). The striatal projections to GP and EP were confirmed using HRP histochemistry. Injections of HRP into GP resulted in the appearance of reactive perikarya which were localized mainly in the central core region of the striatum. HRP injections into EP also produced labeled cells in the striatum but those were found in different regions of the striatum in different animals. It was not possible to discern topograhical relationships.
SINCE the discovery of the endogenous opioid peptides Leu- and Met-enkephalin in mammalian brain, there has been considerable interest in their possible physiological functions1-3. Recent immunohistochemical studies have shown that the enkephalins are localised in neurones in various regions of the central nervous system, with high concentrations in nerve terminals4,5. These morphological studies and the results of detailed regional analyses of rat brain by radioimmunoassay have indicated that enkephalin-containing nerve terminals are particularly concentrated in the rat globus pallidus, which by radioimmunoassay has been found to contain a concentration of enkephalin six to eight times higher than that observed in any other brain region6,7. Here, we describe a calcium-dependent evoked release of enkephalin-like immunoreactivity from slices of rat globus pallidus in vitro. This finding supports the view that enkephalins may be released as neurotransmitter substances from nerve terminals in the brain.
WE have suggested previously that Met-enkephalin and
Leu-enkephalin1 may act as neurotransmitters in the central
and peripheral nervous systems2,3. Recent studies on the
distribution of the enkephalins support this view4-6. An
important step in establishing a possible neurotransmitter role of the
enkephalins is the demonstration of their release from neuronal sites by
potassium ions or veratridine. Indirect evidence for the release of
enkephalin or endorphin has been provided by several
workers7-10. We now present direct evidence for such release
from isolated brain slices and synaptosomal preparations.
THE pentapeptides leucine (Leu-) enkephalin and methionine (Met-)
enkephalin were isolated from brain and shown to be endogenous
opiates1. These peptides bind to the brain opiate
receptor2, and their biological actions are blocked by the
morphine antagonist naloxone3. The availability of synthetic
enkephalins has led to the development of radioimmunoassays (refs 2, 4,
and R. Miller, personal communication) and immunohistochemical
techniques. Preliminary immunohistochemical studies5,6 have
shown the distribution of enkephalin-containing fibres to coincide with
the regional distribution of opiate receptors as determined by
biochemical techniques and autoradiography6-8. The highest
density of enkephalin-positive fibres6,8 was located in the
globus pallidus. We have now confirmed the presence of
enkephalin-immunoreactive fibres in the globus pallidus, and using
specific lesions have shown this immunoreactivity to be abolished by
destruction of the neural connections between the globus pallidus and
the caudoputamen.
Several studies have attempted to determine whether BDZ receptors are localized on glial or neuronal cells. These studies suggest that BDZ receptors in the CNS might be associated with neuronal membranes. Another line of research into the understanding of the location of BDZ receptors in the CNS is the use of postmortem brain tissue from patients dying of neurological disorders. In Huntington's disease (HD), there is selective loss of small interneurons in the basal ganglia as well as widespread neuronal destruction in the cerebral cortex. There is no depletion of glial cell population in these brain areas but rather a proliferation. Several studies have correlated the decrease in various neurotransmitter receptor densities in the basal ganglia and frontal cortex of HD brains with the neuronal degeneration in these regions. To this end, the authors have monitored BDZ receptors in several regions of HD brains to provide further support for the hypothesis that there is a partial population of BDZ receptors on neuronal cell membranes in the brain.
Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [3H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [3H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.
THE idea that morphine and other narcotic analgesics as well as opioid peptides affect the activity of nigrostriatal dopaminergic neurones is well supported. For instance these substances elicit changes in motor activity similar to those observed after blockade of dopamine (DA) receptors by neuroleptics, they increase DA turnover in striatum and several typical symptoms of the abstinence syndrome they provoke involve dopaminergic systems (see refs 1-3 for reviews). In agreement with these observations, a high density of `opiate receptors'4 and of enkephalin binding sites5,6, a high content in enkephalins7 and a dense network of enkephalin-containing neurones8 are found in the striatum. All these observations suggested the possible connection between alleged enkephalinergic neurones and the dopaminergic fibres ending in the striatum. We report here that both the regional distribution of high affinity (morphine-displaceable) binding sites for leucine-enkephalin (Leuenkephalin) in striatum and their decreased number after chemical or mechanical interruption of the nigrostriatal bundle support the assumption that enkephalinergic neurones terminate presynaptically on dopaminergic nerve terminals in the striatum.
Opiate receptor distribution, determined by the autoradiographic localization of stereospecific [3H]diprenorphine binding sites, was examined in the telencephalon. Areas showing very dense or dense localization of receptors included parts of the presubiculum and amygdala, patchy areas in the caudate-putamen and accumbens, the subfornical organ, the interstriatal nucleus of the striae terminalis and the anterior olfactory nucleus, pars externa. Lower densities were found in other parts of the hippocampal formation, the deeper part of the cerebral cortex, the entopeduncular nucleus, globus pallidus, nucleus triangularis septi and nucleus paratenialis. The significance of these findings is discussed in terms of the biochemical and physiological actions of opiates.
3H-GABA binding was measured in the caudate nucleus, putamen, parietal cortex and cerebellar cortex of control patients and patients with Huntington's chorea. The density of 3H-GABA binding in the parietal cortex was similar in both patient groups. In the striatal regions there was a significant large (70–80 percent) decrease in the density of GABA binding which is consistent with the severe atrophy and cell loss observed in these regions. In contrast the membranes prepared from the cerebellar cortex exhibited both an increased binding capacity (at 25 nM 3H-GABA) and an apparently increased affinity (decreased KD) for 3H-GABA. The decreased capacity of the striatum to bind 3H-GABA may partially explain the reported lack of clinical efficacy of GABAmimetic compounds in Huntington's chorea.
The opiate etorphine depresses monosynaptic excitatory postsynaptic potentials (EPSP's) elicited in spinal cord cells by activation
of dorsal root ganglion cells in murine neuronal cell culture. The depression is reversed by naloxone. Statistical analysis
of the synaptic responses reveals that the opiate reduces EPSP quantal content at this synapse without altering quantal size.
Therefore, the opiate action is presynaptic and affects transmitter release rather than postsynaptic responsiveness.
Sensory neurons grown in dispersed cell culture in the absence of non-neuronal cell types contain immunoreactive substance P that is chemically similar to synthetic substance P. When depolarized in high-K+ media (30-120 mM), the neurons release this peptide by a Ca2+-dependent mechanism. An enkephalin analogue, [D-Ala2]enkephalin amide, at 10 micron inhibits the K+-evoked release of substance P. At the same or lower concentrations, [D-Ala2]enkephalin amide and enkephalin decrease the duration of the Ca2+ action potential evoked and recorded in dorsal root ganglion cell bodies without affecting the resting membrane potential or resting membrane conductance. This modulation of voltage-sensitive channels may account for the inhibition of substance P release.
The topographical projections of substance P pathways from the caudateputamen and globus pallidus to the pars compacta and pars reticulata of the substantia nigra have been investigated in the rat using immunohistochemical and radioimmunoassay techniques and compared with the projections of GABA nergic striatal neurones. Unilateral vertical knife cuts through the anterior and posterior striatum have shown the majority of substance P-containing neurones which project to the substantia nigra to originate in the most rostral part of the caudate-putamen. This projection appears to innervate the pars reticulata and pars compacta of the substantia nigra to a similar extent. A separate projection of substance P-containing neurones to the substantia nigra appears to originate in the globus pallidus. Undercutting the cerebral cortex which overlies the corpus striatum did not affect the substance P content of the globus pallidus or substantia nigra. However, there appears to be an additional substance P projection from the basal ganglia to the entopeduncular nucleus. In contrast, GABA-containing neurones which project to the substantia nigra are mainly located in more caudal parts of the caudate-putamen and in the globus pallidus. There is a marked differentiation in the region of the substantia nigra innervated by GABA cells originating in the rostral and caudal parts of the corpus striatum. Rostrally situated neurones project almost exclusively to the pars reticulata, while neurones in the caudal part of the caudate-putamen and globus pallidus project to both the pars compacta and pars reticulata. These results suggest that there is a partial topographical separation of the sites of origin of substance P- and GABA-containing neurones which project to the substantia nigra.
The topographical distribution of glutamate decarboxylase (GAD), aromatic amino acid decarboxylase (AAD) and choline acetyltransferase (ChAT) were studied in striatum (i.e. caudate-putamen), globus pallidus, nucleus entopeduncularis and substantia nigra.There were only small differences in the rostrocaudal distribution of enzymes in striatum. The highest concentration of GAD was found in the ventrocaudal part, whereas AAD was highest in the rostral part. ChAT did not show any distinct distribution pattern. Globus pallidus and nucleus entopeduncularis were similar in their content of GAD, AAD and ChAT. They were rich in GAD but poor in AAD and ChAT. The highest concentration of GAD was found in the rostralmost part of globus pallidus. In substantia nigra AAD was concentrated in the rostral part; contents of GAD and ChAT did not differ distinctly in the rostral and caudal nigra.GAD and ChAT were highly localized in a particulate fraction, probably in all 4 regions, whereas AAD was localized in particulate fraction in the striatum and globus pallidus and soluble in the nucleus entopeduncularis and substantia nigra.Transverse and/or oblique hemitransections which passed through the striatum but not through the globus pallidus resulted in substantial loss of GAD in the globus pallidus, nucleus entopeduncularis and substantia nigra. There was a good correlation between the anteroposterior level of hemitransection and the decline in GAD activities, which was generally highest following posterior lesions. The reduction of GAD was largest in the globus pallidus and smallest in the substantia nigra, in which a significant loss of GAD occurred only following transections of the postcommissural part of caudate-putamen. A very high decrease of GAD in the nucleus entopeduncularis and substantia nigra was obtained following posterior oblique hemitransections which passed through posterior part of striatum and rostral globus pallidus.The results indicate that the majority of GABAergic terminals in the globus pallidus belong to striatopallidal fibers. They suggest, furthermore, that a large number of striatoentopeduncular and striatonigral fibers are GABAergic, the latter arising preferentially from the posterior part of caudate-putamen.
The regional distribution of leu- and met-enkephalin in the brain of rats killed with a microwave beam focused on the skull was studied using a radioimmunoassay. The enkephalin content varied significantly in the seven regions of rat brain studied: the highest concentration was found in striatum and the lowest in cerebellum and hippocampus. In every brain region studied, the content of met- was higher than that of leuenkephalin. In the brain of rats killed with focused microwave, the content of enkephalins is higher than in brains of rats killed by decapitation. It seems unlikely that this difference is due to an artifact caused by microwave radiation.
In the guinea pig ileum myenteric plexus--longitudinal muscle preparation, dynorphin-(1--13) and the prototypical kappa agonist ethylketocyclazocine had equally poor sensitivity to naloxone antagonism and showed selective cross protection in receptor inactivation experiments with the alkylating antagonist beta-chlornaltrexamine. In binding assays with membranes from guinea pig brain, ethylketocyclazocine and dynorphin-(1--13) amide were more potent in displacing tritium-labeled ethylketocyclazocine than in displacing typical mu and delta opioid receptor ligands. In the two preparations studied, the dynorphin receptor appears to be the same as the kappa opioid receptor.
A study of the anatomical distribution of the endogenous opioid dynorphin in rat brain showed that the peptide is localized in a widespread system with multiple cell groups and projections. This network is revealed by the use of multiple antiserums against dynorphin and can be distinguished from the system containing methionine-enkephalin and leucine-enkephalin, which is mapped by the use of antiserums against the enkephalins and biosynthetically related peptides in the adrenal. It thus appears that the brain contains at least three separate opioid neuronal networks: an enkephalin family with components similar to those found in the adrenal, a beta-endorphin family, and a dynorphin family.
The regional distributions of substance P and Methionine-enkephalin (Met-enkephalin) were determined in normal human brains and in Huntington's disease using sensitive radioimmunoassays. Model experiments showed that both Met-enkephalin- and substance P-like immunoreactivities were stable for up to 72 h post-mortem in mouse brain. The results of high pressure liquid chromatography (HPLC) analyses indicated that the majority of the immunoreactivity detected in human globus pallidus corresponded to the native peptides, substance P or Met-enkephalin. In Huntington's disease the present results confirm that there is a substantial drop (> 80%) in the substance P content of the globus pallidus (both medial and lateral segments) and substantia nigra, and there was also a reduction (> 50%) in the Met-enkephalin content of these areas. This result suggests the loss of striato-pallidal and striato-nigral substance P and enkephalin-containing projections in Huntington's disease.
After degeneration of serotoninergic neurons induced by either transection of the ascending neuronal pathways originating from the nucleus raphe dorsalis or intraventricular 5,6-dihydroxytryptamine administration, the number of binding sites for [3H]D-Ala2, Met5-enkephalinamide was significantly reduced. This decrease in binding sites does not seem to be related to the opiate receptors present on dopaminergic terminals, nor is it due to a simple decrease in serotoninergic neuronal tone, since after p-chlorophenylalanine (100 mg/kg X 4 days) the number of striatal binding sites for the opiate ligand was not diminished. On the other hand, shortly after mechanical interruption of the raphe-striatal serotoninergic fibers, at a time when the metabolic processes are still functioning in the lesioned neurons, morphine still increased the striatal content of 5-hydroxyindoleacetic acid. These results suggest the presence of opiate receptors on striatal serotoninergic terminals, where they may modulate the presynaptic activity of these neurons.
Radioautography of tritiated diprenorphine in rat brain indicates anatomic distribution of receptors with a greater degree of precision than is possible using dissection techniques. The results of this study largely confirm those of others but indicate some differences in receptor distribution in the thalamus. Differential receptor binding in the periaquaductal gray matter with the highest counts lying laterally is an original observation.
Autoradiographic localization of opiate receptors in the rat striatum with specifically bound 3H-diprenorphine reveals relatively small, high density clusters of receptors, a high density band of receptors along the striatal-callosal border, and a lower density of receptors spread over the remainder of the neuropil. Placement of kainate lesions resulted in a 94% loss of receptors in the clusters and a smaller loss in other areas. Removal of the dopamine-containing input to the striatum by placement of medial forebrain bundle lesions or by intrastriatal injection of 6-hydroxydopamine resulted in a greater depletion of receptors in the non-cluster areas compared to cluster areas. In repeated biochemical and autoradiographic studies, no change was found after either decortication or placement of thalamic lesions. It is concluded that the bulk of striatal opiate receptors are localized postsynaptically on intrinsic striatal neurons and their processes with the bulk of the remaining receptors localized to the dopamine-containing axons and terminals.
In order to obtain information on the possible functions of endogenous opiates in the primate cerebral cortex, we assessed the distribution of mu-like opiate receptors (which selectively bind 3H-labeled naloxone) and delta-like opiate receptors (which selectively bind 3H-labeled D-Ala2, D-Leu5-enkephalin) throughout the cerebral cortex of the rhesus monkey. Stereospecific [3H]naloxone binding sites increased in a gradient along hierarchically organized cortical systems that sequentially process modality-specific sensory information of a progressively more complex nature. Specific [3H]enkephalin binding sites, in contrast, were relatively evenly distributed throughout the cerebral cortex. These results, in combination with electrophysiological studies of monkeys and humans, suggest that mu-like opiate receptors may play a role in the affective filtering of sensory stimuli at the cortical level, that is, in emotion-induced selective attention.
GABA levels, high affinity GABA uptake and glutamic acid decarboxylase levels are reduced in rat ventroanterolateral thalamic nucleus after destruction of the entopeduncular nucleus with kainic acid. This is strong evidence that GABA is an entopedunculothalamic neurotransmitter. The striatoentopeduncular pathway is also GABAergic. Thus the function of the corpus striatum may be to disinhibit the thalamus.
The synthetic peptide NH2-Tyr-Pro-Phe-Pro-CONH2 (morphiceptin), which is the amide of a fragment of the milk protein beta-casein,
has morphinelike activities and is highly specific for morphine (mu) receptors but not for enkephalin (delta) receptors. It
is as active as morphine in the guinea pig ileum but much less active in the mouse and rat vas deferens. The discovery of
this specific morphine receptor ligand substantiates the hypothesis of multiple opiate receptors. The ligand, which may be
of physiological significance since a very similar, or identical, activity can be detected in enzymatic digests of beta-casein,
may prove useful for further investigation of the functions of opiate receptor subtypes.
The origin of enkephalin-immunoreactive nerve terminals in the globus pallidus was investigated by combining immunocytochemistry with stereotaxic injection of neurotoxic agents (colchicine and kainic acid) and microknife deafferentations. The intracerebral administration of colchicine, irrespective of whether in the caudate putamen or in globus pallidus, induces the appearance of enkephalin-immunoreactive cell bodies and fibres in the caudate putamen. No immunoreactive cell bodies were depicted in the globus pallidus after this treatment. Kainic acid injections in the caudate putamen produced topographic depletions of enkephalin-immunoreactive terminals in the globus pallidus. The more anterior injections produced medial-anterior depletions, while posterior injections gave latero-posterior depletions. Injections in the globus pallidus produced only a non-specific loss of fluorescence restricted to the tip of the cannula. Coronal microknife cuts produced a combination of build-up and depletion of enkephalin immunofluorescence according to the position of the cut. The build-up of immunoreactive materials was always observed in the caudate-putamen side of the cut while depletions observed in the globus pallidus were related to the extent of deafferentation of this nucleus from the caudate putamen. All these observations confirmed the neostriatal origin (caudate putamen) of the enkephalinergic fibres present in the paleostriatum (globus pallidus).
By employing both in vivo and in vitro labeling techniques, opiate receptors were labeled with tritiated diprenorphine in the monkey brain and localized by light microscopic autoradiography. Both methods of labeling gave similar results, allowing a description of discrete areas having opiate binding sites. High concentrations of opiate receptors were found in the substantia gelatinosa of the spinal cord, nucleus tractus solitarius, area postrema, lateral parabrachial nucleus, substantia grisea centralis, several nuclei of the thalamus and hypothalamus, substantia innominata and in the amygdala. In the monkey pituitary, receptors were found in the neurohypophysis. These results correlate well with those found in autoradiographic studies of the rat brain although there are a few notable differences. Many of the opiate receptor distributions can be correlated with anatomical loci of brain functions known to be influenced by administration of opiate compounds.
Rats received lesions of either the cortico-striatal or the nigrostriatal projections. Two to three days after either type of lesion, evidence for degenerating dendritic spines that were postsynaptic to degenerating striatal boutons was often encountered. This type of transsynaptic degeneration of spines indicates that it is inappropriate to conclude that decreases in the number of neurotransmitter receptors after these types of lesions necessarily indicates that these receptors are located presynaptically on striatal afferents.
We studied gamma-aminobutyric acid (GABA), benzodiazepine, and muscarinic cholinergic receptor-binding by quantitative autoradiography. In coronal sections from the brain of a patient with Huntington disease, binding for all three receptors in caudate and putamen was lower than control values. Binding to GABA and benzodiazepine receptors was increased in lateral and medial pallidum and decreased in ventrolateral thalamus. Muscarinic cholinergic receptors were markedly decreased in pallidum but not thalamus. The findings suggest that loss of striatal afferents to both segments of pallidum results in GABA and benzodiazepine receptor supersensitivity, and support the utility of quantitative autoradiography for receptor studies in human postmortem material.
We have investigated the effect of nigral 6-hydroxydopamine (6-OHDA) lesions on binding of the mu receptor ligand dihydromorphine (DHM) and the delta receptor ligand [D-Ala2, D-Leu5]-enkephalin (DADLE) to sections of rat striatum under conditions which yield mu-like and delta-like ligand selectivities at discrete receptor patches (Type 1 receptor). 3H-DHM binding was decreased 43% while 3H-DADLE was decreased 22%. However, when the contribution of diffuse binding (Type 2) which is not affected by 6-OHDA is subtracted from the patch, the decrease is approximately 49% for both ligands. These data support the hypothesis that the Type 1 receptor of striatal patches is a conformationally malleable receptor entity which can exist in states having high affinities for various classes of opiate ligands.
The binding of a radiolabeled opiate agonist ([3H]etorphine) and antagonist ([3H]naloxone) was studied using quantitative film autoradiography of rat-brain sections labeled by in vitro dipping methods. The binding activities of both [3H]naloxone and [3H] etorphine were saturable in three brain regions: noncluster striatum, nucleus accumbens and cingulate cortex. Eadie-Hofstee analysis of these regions yielded the following binding affinities and capacities: noncluster striatum binding affinity (KD) +/- S.E. = 1.59 +/- 0.23 nM, maximal binding capacity (Bmax) +/- S.E. = 28.3 +/- 1.9 fmol/mg, S.D. error of the raw data (Erad) = 6.4%; nucleus accumbens, KD +/- S.E. = 1.74 +/- 0.28 nM, Bmax +/- S.E. = 73.3 +/- 5.2 fmol/mg, S.D. (Erad) = 6.2%; cingulate cortex, KD +/- S.E. = 1.44 +/- 0.15 nM, Bmax +/- S.E. = 37.6 +/- 1.4 fmol/mg, S.D. (Erad) = 2.5%. A KD +/- S.E. = 1.72 +/- 0.29 nM, Bmax +/- S.E. = 74.1 +/- 5.3 fmol/mg, S.D. (Erad) = 5.0% was found for [3H]etorphine binding in the noncluster striatum. Hill plots of both [3H]naloxone and [3H]etorphine binding in noncluster striatum demonstrated an absence of cooperativity with slopes of 1.01 and 1.07, respectively. Stereospecificity of binding was confirmed by competition for 2.0 nM [3H]naloxone in the noncluster striatum with a levorphanol IC50 = 5.5 nM and a dextrorphan IC50 greater than 1000 nM. Rank order potency for competition for 2.0 nM [3H]naloxone binding in noncluster striatum was etorphine greater than naloxone greater than levorphanol greater than morphine greater than dextrorphan. The regional order of binding activities (femtomoles per milligram +/- S.D.) for 2.0 nM [3H]naloxone was as follows: striatal clusters (111.1 +/- 24.5) greater than interpeduncular nucleus (77.8 +/- 10.1) greater than central nucleus of amygdala (64.5 +/- 9.7) greater than nucleus accumbens (34.4 +/- 6.9) greater than median raphe (24.4 +/- 6.1) greater than striatal noncluster (23.3 +/- 3.5) greater than superior colliculus striatum grieseum (22.2 +/- 4.0). Thus, quantitative film autoradiography of brain sections labeled in vitro may be used to characterize the pharmacological binding properties of ligands in many small brain regions not amendable to study in membrane preparations.
Opioid peptide-like (OPL)-immunoreactivity and (the GABA-biosynthetic enzyme) glutamic acid decarboxylase-like (GAD)-immunoreactivity were localized in rat neostriatum and central amygdaloid nucleus (ACE) using a polyclonal sheep antiserum to rat brain GAD and a monoclonal mouse antibody to the N-terminus of beta-endorphin (3-E7) as primary antisera. PAP-immunohistochemistry revealed GAD-immunoreactivity in the majority of neurons in neostriatum and ACE. OPL-immunoreactivity was observed in numerous neurons in ACE, but only in few neostriatal nerve cells. In double immunofluorescence in the same section OPL- and GAD-immunoreactivity colocalized in few medium size cells in the neostriatum, but in numerous neurons in ACE. The existence of opioid peptide containing GABAergic neurons in ACE and neostriatum is demonstrated.
Area measurements taken from receptor autoradiograms were employed to estimate the size of striatal kainate lesions and the amount of shrinkage in deafferented projection areas. There was no significant difference in the size of substantia nigra (SN) on the denervated side as compared to the intact side one week and one month after unilateral striatal lesions. Although there was no change in the size of globus pallidus (GP) on the lesioned side one week after the lesion, there was a 17% shrinkage one month after the lesion. At 3-4 months after the lesion, the amount of shrinkage was 19% in SN and 16% in GP.