Modification of d-amphetamine- or chlorpromazine-induced hypothermia by β-endorphin,MIF-I,and α-MSH: Mediation by the dopaminergic system

The effects of β-endorphin, MIF-I, and α-MSH on d-amphetamine- and CPZ-induced hypothermias in rats kept at 4°C were tested in three experimental groups: (a) intact; (b) rats with lesions of the olfactory tubercle; and (c) rats in which the link between the DA mesolimbic pathway and the striatum was disconnected. All drugs tested alone (except MIF-I) caused significant hypothermia. Pretreatment with CPZ, MIF-I, and α-MSH potentiated d-amphetamine-induced hypothermia in intact rats. Pretreatment with α-MSH potentiated CPZ-induced hypothermia. β-Endorphin partially blocked d-amphetamine-induced hypothermia, but did not interact with CPZ, MIF-I, or α-MSH. All potentiations were either reduced or disappeared in the incisioned rats. CPZ and α-MSH caused hypothermia in olfactory tubercle-lesioned rats. The results indicate that: (a) the DA mesolimbic pathway is involved in the hypothermic response of all drugs tested; (b) an intact feedback loop is required for the potentiation of the hypothermic response of CPZ on d-amphetamine, MIF-I on d-amphetamine, and α-MSH on d-amphetamine and CPZ; (c) β-endorphin acts as a partial blocker of d-amphetamine; MIF-I is a weak potentiator of d-amphetamine. α-MSH acts as a negative modulator of the DA system, most probably in the striatum.     

Interstitial 3-Methoxytyramine Reflects Striatal Dopamine Release: An In Vivo Microdialysis Study

Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3-methoxytyramine (3-MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3-MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3-MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3-MT. Increases in DA and 3-MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3-MT in the dialysate, gamma-Butyrolactone also greatly decreased DA and 3-MT. Finally, d-amphetamine produced a large increase in DA and 3-MT in animals that had been treated previously with gamma-butyrolactone. The Pearson correlation coefficients for DA and 3-MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3-MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3-MT may not reliably reflect DA release following certain pharmacological manipulations.     

Amphetamine-induced inhibition of central noradrenergic neurons: a pharmacological analysis

The amphetamine-induced inhibition of brain noradrenaline (NA) containing neurons in the rat locus coeruleus (LC) was pharmacologically analyzed utilizing single unit recording techniques. The presynaptic α-receptor blocking agent yohimbine (10 mg/kg i.p., 30 min before) largely prevented the amphetamine-induced depression of LC units in contrast to prazosin (0.6 mg/kg i.p., 30 min) or phenoxybenzamine (20 mg/kg, 30 min) which both slow preference for postsynaptic α-receptors. The β-receptor blocking agent, propranolol (10 mg/kg, 30 min), as well as the peripherally but not centrally active α-receptor blocking drug phentolamine (10 mg/kg, i.p., 30 min), also did not block the amphetamine effect. The LC inhibition by amphetamine was blocked by pretreatment with reserpine (10 mg/kg, i.p., 5 h), which caused almost total depletion of brain catecholamines. However, unlike the amphetamine-induced inhibition of central dopamine (DA) neurons the NA cell inhibition was not blocked by pretreatment with a tyrosine hydroxylase inhibitor (α-MT, 50 or 250 mg/kg i.p., 30 min). These results suggest that the amphetamine-induced inhibition of NA neurons in the LC is an indirect effect, mediated via activation of central α-receptors of presynaptic character. The lack of antagonism by α-MT indicate that the NA release by amphetamine, unlike its effect on brain DA, is not critically dependent on the rate of tyrosine hydroxylation. Thus the euphoriant action of amphetamine, which is blocked by α-MT, may be associated with release of DA rather than NA in brain.     

beta-Endorphin administration increases hippocampal acetylcholine levels.

The contents of acetylcholine and choline were determined in rat cortex, striatum, and hippocampus following intraventricular injection of β-endorphin or D-Ala²-enkephalinamide, a synthetic enkephalin analog, in doses known to produce analgesia in experimental animals. These opiate polypeptides produced significant increases in acetylcholine levels in the hippocampus, a subcortical structure rich in cholinergic terminals. The acetylcholine content of the hippocampus (but not the cortex or striatum) was significantly elevated 15, 30, and 60 minutes after a single intraventricular injection of β-endorphin (10 μg/brain) or D-Ala²-enkephalinamide (10 μg/brain). Peak alterations in regional acetylcholine concentrations and in analgetic effectiveness both occurred 30 minutes after peptide administration. Choline concentrations were unchanged by any of the experimental treatments. Naloxone hydrochloride (1 mg/kg, subcutaneously) affected neither brain acetylcholine concentrations, nor the response latencies of rats placed on a hot-plate; it did, however, antagonize the changes in these parameters caused by β-endorphin or D-Ala²-enkephalinamide. These data suggest that endorphins may normally regulate the physiologic activity of some cholinergic neurons.     

Endorphins and the central inhibition of urinary bladder motility

The involvement of endogenous opioid mechanisms in the central neurogenic control of urinary bladder function has been examined in anesthetized rats. Intracerebroventricular (ICV) microinjection of β-endorphin (0.5–2.0 μg) produced powerful inhibition of rhythmic bladder contractions initiated by central reflex activity. The peptide fragments γ-endorphin and α-endorphin (4–16 μg), formed by the processing of β-endorphin by membrane homogenates of brain, were less active than the parent compound. The inhibitory effects of β-endorphin was reversed by ICV naloxone (1–2 μg) but higher doses were required to reverse γ- or α-endorphin effects. ICV naloxone administered alone increased intravesicular pressure and bladder contraction frequency. These observations support the hypothesis that the endorphins have a physiological role in the central regulation of urinary bladder activity.     

The influence of beta-LPH fragments on alpha-MSH release: the involvement of a dopaminergic system

β-Endorphin was able to enhance plasma α-MSH levels in rats after intracerebroventricular injection. This effect could be inhibited by naloxone or by removing tyrosine from position 61 of the peptide. Neither α- and γ-endorphin nor their des-tyrosine analogs appeared to be able to modify plasma α-MSH levels. The stimulating effect of β-endorphin on plasma α-MSH levels could be completely blocked by a simultaneous injection of apomorphine, in an amount in which apomorphine itself had no effect on α-MSH levels in plasma. A single injection of haloperidol increased plasma α-MSH levels in a dose related manner. A dose of haloperidol, which caused an apomorphine antagonizable increase in plasma α-MSH, did not modify β-endorphin elevated α-MSH levels. A high concentration of haloperidol was able to stimulate the basal release of α-MSH from isolated pituitaries $\text{in bitro}$, whereas β-endorphin appeared to be inactive in this respect.These observations indicate a central opiate receptor-mediated influence of β-endorphin on α-MSH release and the possible involvement of a dopaminergic system, mediating the β-endorphin effect.     

β-endorphin: Dose-dependent suppression of fixed-ratio operant behavior

Centrally administered β-endorphin or morphine suppressed fixed-ratio 15, food-reinforced responding by rats in a dose- dependent manner. β-Endorphin was 21 times more potent than morphine on a molar basis. Scratching and wet-dog shakes were observed within 30 minutes of β-endorphin administration but were not seen after morphine and did not appear to be responsible for the suppression of the conditioned behavior.     

Halothane attenuated haloperidol and enhanced clozapine-induced dopamine release in the rat striatum

The effect of halothane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA)) induced by neuroleptics was studied using in vivo microdialysis techniques. Halothane attenuated haloperidol-induced dopamine release and enhanced clozapine-induced dopamine release in the rat striatum.A microdialysis probe was implanted into the right striatum of male SD rats. Rats were given saline or the same volume of 200 microg kg(-1) haloperidol (D(2) receptor antagonist), 10 mg kg(-1) sulpiride (D(2) and D(3) antagonist), or 10 mg kg(-1) clozapine (D(4) and 5-HT(2) antagonist) intraperitoneally with or without 1-h halothane anesthesia (0.5 or 1.5%). Halothane anesthesia did not change the extracellular concentration of DA, but increased the metabolite concentrations in a dose-dependent manner. The increased DA concentration induced by haloperidol was significantly attenuated by halothane anesthesia, whereas the metabolite concentrations were unaffected. Halothane had no effect on the changes in the concentrations of DA or its metabolites induced by sulpiride. The clozapine-induced increases in DA and its metabolites were enhanced by halothane anesthesia.Our results suggest that halothane anesthesia modifies the DA release modulated by antipsychotic drugs in different ways, depending on the effects of dopaminergic or serotonergic pathways.     

Stress-Related Changes in Cerebral Catecholamine and Indoleamine Metabolism: Lack of Effect of Adrenalectomy and Corticosterone

The concentrations of catecholamine and indoleamine metabolites were measured in intact and adrenalectomized mice to determine whether adrenal hormones mediate or modulate the stress-induced responses. Thirty minutes of footshock resulted in significant increases of the ratios of the dopamine (DA) catabolite, dihydroxyphenylacetic acid (DOPAC), to DA in prefrontal cortex, nucleus accumbens, striatum, hypothalamus, and brainstem, and of homovanillic (HVA)/DA ratios in nucleus accumbens, striatum, amygdala, and hypothalamus. Ratios of 3-methoxy-4-hydroxyphenylethyleneglycol to norepinephrine (NE) were also increased in prefrontal cortex, nucleus accumbens, septum, amygdala, hypothalamus, hippocampus, and brainstem. The concentration of NE was decreased in amygdala. 5-Hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT, serotonin) ratios and free tryptophan were also increased in every brain region. Very similar data were obtained from mice restrained for 30 min. Adrenalectomy resulted in increased HVA/DA ratios in prefrontal cortex and striatum, and 5-HIAA/5-HT in septum. The stress-related changes were largely similar in adrenalectomized mice. Significant interactions between adrenalectomy and footshock treatment occurred in prefrontal cortical DOPAC/DA and hypothalamic NE which was depleted only in adrenalectomized mice, suggesting tendencies for these measures to be more responsive in adrenalectomized mice. Corticosterone administration (0.5-2.0 mg/kg s.c.) which resulted in plasma concentrations in the physiological range did not alter the concentrations of the cerebral metabolites measured in any region. We conclude that adrenal hormones do not mediate cerebral catecholamine or indoleamine metabolism in stress, although adrenalectomy may affect HVA and 5-HIAA metabolism, and there was a tendency for catecholamines to be more sensitive to stress in adrenalectomized animals.     

Local Influence of Endogenous Norepinephrine on Extracellular Dopamine in Rat Medial Prefrontal Cortex

Abstract: Noradrenergic and dopaminergic projections converge in the medial prefrontal cortex and there is evidence of an interaction between dopamine (DA) and norepinephrine (NE) terminals in this region. We have examined the influence of drugs known to alter extracellular NE on extracellular NE and DA in medial prefrontal cortex using in vivo microdialysis. Local application of the NE uptake inhibitor desipramine (1.0 µM) delivered through a microdialysis probe increased extracellular DA (+149%) as well as NE (+201%) in medial prefrontal cortex. Furthermore, desipramine potentiated the tail shock-induced increase in both extracellular DA (stress alone, +64%; stress + desipramine, +584%) and NE (stress alone, +55%; stress + desipramine, +443%). In contrast, local application of desipramine did not affect extracellular DA in striatum, indicating that this drug does not influence DA efflux directly. Local application of the α₂-adrenoceptor antagonist idazoxan (0.1 or 5.0 mM) increased extracellular NE and DA in medial prefrontal cortex. Conversely, the α₂-adrenoceptor agonist clonidine (0.2 mg/kg; i.p.) decreased extracellular NE and DA in medial prefrontal cortex. These results support the hypothesis that NE terminals in medial prefrontal cortex regulate extracellular DA in this region. This regulation may be achieved by mechanisms involving an action of NE on receptors that regulate DA release (heteroreceptor regulation) and/or transport of DA into noradrenergic terminals (heterotransporter regulation).     

3-Methoxytyramine Is the Major Metabolite of Released Dopamine in the Rat Frontal Cortex: Reassessment of the Effects of Antipsychotics on the Dynamics of Dopamine Release and Metabolism in the Frontal Cortex, Nucleus Accumbens, and Striatum by a Simple Two Pool Model

Abstract: 3-Methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC) rates of formation were used, respectively, to assess the dynamics of dopamine (DA) release and turnover in the rat frontal cortex, nucleus accumbens, and striatum. Assuming total (re)uptake and metabolism of released DA are relatively uniform among the three brain regions, a simplified two pool model was used to assess the metabolic fate of released DA. Under basal conditions, 3-MT formation was found to comprise >60% of total DA turnover (sum of 3-MT plus DOPAC rates of formation) in the frontal cortex, and not more than 15% in the nucleus accumbens and striatum. Haloperidol increased the 3-MT rate of formation to a greater extent in the frontal cortex than in the two other regions. Clozapine increased the 3-MT rate of formation in the frontal cortex and decreased it in the striatum. Both drugs increased DOPAC rate of formation in the frontal cortex and nucleus accumbens. It was elevated by haloperidol but not clozapine in the striatum. It is concluded that (1) O -methylation is a prominent step in the catabolism of DA in the frontal cortex under both physiological conditions and after acute treatment with antipsychotics, (2) 3-MT is the major metabolite of released DA in the frontal cortex and possibly also in the nucleus accumbens and striatum, (3) in contrast to the frontal cortex, most of the DOPAC in the nucleus accumbens and striatum appear to originate from intraneuronal deamination of DA that has not been released, (4) because presynaptic uptake and metabolism of DA give rise to DOPAC, whereas postsynaptic uptake and metabolism produced both DOPAC and 3-MT, the ratio of 3-MT to DOPAC rates of formation can be a useful index of reuptake inhibition.     

The effects of Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) on ischemia-induced changes of brain acetylcholine and monoamine levels in gerbils

The changes in acetylcholine (ACh), monoamine and monoamine metabolite levels following cerebral ischemia in Mongolian gerbils were examined. In addition, the effects of Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960), which is a spray-dried mixture of 9 herbal drugs, on these changes were also examined. The dramatic decrement of ACh levels in ischemic gerbils was significantly inhibited by p.o. administration of TJ-960 at a daily dose of 3.5 g/kg or 700 mg/kg for one month. Norepinephrine (NE) was also reduced in all ischemic brain regions, and TJ-960 also recovered the level of NE. In ischemic gerbil brains, the dopamine (DA) levels decreased and its metabolites increased in the striatum, but DA and its metabolites in the thalamus+midbrain region increased. The serotonin (5HT) level was reduced in the cerebral cortex and hippocampus. TJ-960 inhibited these monoaminergic changes in ischemic gerbils. This suggests that TJ-960 may provide anti-ischemic action and beneficial effects on various symptoms induced by ischemia.     

Amphetamine-Induced Dopamine Release in the Rat Striatum: An In Vivo Microdialysis Study

The effects of a number of biochemical and pharmacological manipulations on amphetamine (AMPH)-induced alterations in dopamine (DA) release and metabolism were examined in the rat striatum using the in vivo brain microdialysis method. Basal striatal dialysate concentrations were: DA, 7 nM; dihydroxyphenylacetic acid (DOPAC), 850 nM; homovanillic acid (HVA), 500 nM; 5-hydroxyindoleacetic acid (5-HIAA), 300 nM; and 3-methoxytyramine (3-MT), 3 nM. Intraperitoneal injection of AMPH (4 mg/kg) induced a substantial increase in DA efflux, which attained its maximum response 20-40 min after drug injection. On the other hand, DOPAC and HVA efflux declined following AMPH. The DA response, but not those of DOPAC and HVA, was dose dependent within the range of AMPH tested (2-16 mg/kg). High doses of AMPH (greater than 8 mg/kg) also decreased 5-HIAA and increased 3-MT efflux. Depletion of vesicular stores of DA using reserpine did not affect significantly AMPH-induced dopamine efflux. In contrast, prior inhibition of catecholamine synthesis, using alpha-methyl-p-tyrosine, proved to be an effective inhibitor of AMPH-evoked DA release (less than 35% of control). Moreover, the DA releasing action of AMPH was facilitated in pargyline-pretreated animals (220% of control). These data suggest that AMPH releases preferentially a newly synthesised pool of DA. Nomifensine, a DA uptake inhibitor, was an effective inhibitor of AMPH-induced DA efflux (18% of control). On the other hand, this action of AMPH was facilitated by veratrine and ouabain (200-210% of control). These results suggest that the membrane DA carrier may be involved in the actions of AMPH on DA efflux.     

beta-Endorphin concentration in the brain of intact and hypophysectomized rats.

The brain concentration and distribution of β-endorphin immunoreactivity in the brain have been studied in intact and hypophysectomized rats. The results obtained with different methods for killing the animals and extracting β-endorphin are compared. Different methodologies of killing the rat and extracting the brain yield concentrations of β-endorphin which vary ten fold. Consistently the highest concentrations of β-endorphin have been found in the hypothalamus, midbrain and hindbrain. After hypophysectomy major reduction of β-endorphin concentration in the brain was observed.     

α-methyl-6-aminodopamine: Depletion of catecholamines in mouse brain and peripheral tissues

A new synthetic agent R, S-2-amino-1(2-amino-4, 5-dihydroxyphenyl) propane dihydrobromide, also referred to as α-methyl-6-aminodopamine (α-Me-6-ADA), has been found to produce acute (one day) and longer-term (seven day) depletion of norepinephrine (NE) levels in mouse brain and peripheral tissues. A 100 mg/kg dose of α-Me-6-ADA (i.v., free base) produced greater than 85% depletion of NE in the heart and spleen at one day and one week after treatment. Intracranially, α-Me-6-ADA (100 μg i.vtr.) depleted NE in the telencephalon and brain stem by 79% and 21% respectively at seven days. In addition DA was depleted by 45% in the ipsilateral striatum. The α-Me-6-ADA appears to have a relative selectivity for noradrenergic nerves, as an intracranial dose of 10 μg, which decreased NE in mouse whole brain by 52% at one day, failed to alter the DA content. These data suggest that α-Me-6-ADA may be a neurotoxin.     

Effects of beta-endorphin fragments on plasma levels of vasopressin

Plasma vasopressin levels are significantly decreased after intracerebroventricular (icv) administration of β-endorphin (βE), but not of des-tyrosine βE (DTβE). The βE induced decrease of vasopressin levels, which occurs in normal as well as in water deprivated rats, can partially be blocked by naltrexone. γ-Endorphin (γE), α-endorphin (αE), DTγE and DTαE did not affect basal levels of vasopressin, but γE and DTγE further increased the elevated vasopressin levels in water deprivated rats. Naltrexone antagonized this increase following γE administration, but not that induced by DTγE. The results suggest that the effects of βE and its fragments on plasma vasopressin levels are mediated by multiple opiate and non-opiate receptor systems.     

Intracerebral dialysis: direct evidence for the utility of 3-MT measurements as an index of dopamine release

Intracerebral dialysis was used to monitor the in vivo efflux of striatal dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and 3-methoxytyramine (3-MT) in the pentobarbital anesthetized rat. In untreated rats, there were low levels of extra-cellular DA and 3-MT which were increased 15-fold by treatment with amphetamine. Under basal and drug-stimulated conditions, 3-MT concentrations were maintained at approximately 30% of the extracellular DA levels. These data agree with in vivo turnover estimates which indicate that 20 to 30% of DA turnover is through the 3-MT pool in the striatum. In contrast, extracellular DOPAC and HVA levels were reduced only slightly by amphetamine and with a delayed onset. Our data support the hypothesis that striatal DOPAC is an accurate index of intraneuronal DA metabolism and that 3-MT is an index of the extracellular concentration of DA.     

Effect of amantadine on the rate of dopamine synthesis in rat corpus striatum.

Abstract— The effect of amantadine on the rate of dopamine synthesis in rat corpus striatum was determined by three methods. (1) Measuring the rate of decline of endogenous dopamine following inhibition of synthesis with a-methyltyrosine (α-MT); (2) Measuring the rate of conversion of [3,5-³H]tyrosine to ³H-labelled catechols under conditions of an initial rate; and (3) measuring the levels of homovanillic acid (HVA), the principal metabolite of brain dopamine. Endogenous dopamine levels were 68-1 n-mole/g with a control synthesis rate of about 21 n-mole/g/h as determined using either α-MT or [3,5-³H]tyrosine. Amantadine had no effect on synthesis at doses up to 100 mg/kg using α-MT and [3,5-³H]tyrosine. HVA levels were unaffected after 30 mg/kg drug, but were elevated 48%(P < 005) after 100 mg/kg of drug. By contrast apomorphine reduced and haloperidol increased synthesis as determined by all three methods. It is concluded that amantadine has no marked effect on dopamine synthesis in rat corpus striatum.     

Relationship Between Plasma and Cerebrospinal Fluid Norepinephrine and Dopamine Metabolites in a Nonhuman Primate

Major and minor pathways of metabolism in the mammalian CNS result in the formation of 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and normetanephrine (NMN) from norepinephrine (NE), and homovanillic acid (HVA) and 3-methoxytyramine (3-MT) from dopamine (DA), respectively. The correlational relationships between HVA and 3-MT and between MHPG and NMN in primate CSF and plasma have not been described. These relationships may help to elucidate the usefulness of CSF and plasma metabolites as indices of CNS NE and DA activity. In addition, because NMN is unlikely to cross the blood-brain barrier. CSF NMN concentrations would not be confounded by contributions from plasma, which is a major issue with CSF MHPG. We have obtained repeated samples of plasma and CSF from drug-naive male squirrel monkeys and have measured the concentrations of MHPG, HVA, NMN, and 3-MT to define their correlational relationships. For the NE metabolites, significant correlations were obtained for CSF MHPG and NMN (r = 0.806, p less than 0.001), plasma MHPG and CSF NMN (r = 0.753, p less than 0.001), and plasma and CSF MHPG (r = 0.776, p less than 0.001). These results suggest that CSF and plasma MHPG and CSF NMN may reflect gross changes in whole brain steady-state noradrenergic metabolism. Only a single significant relationship was demonstrated for the DA metabolites, with CSF 3-MT correlating with plasma HVA (r = 0.301, p less than 0.025). The results for the DA metabolites probably reflect regional differences in steady-state brain dopaminergic metabolism.     

Effects of l-DOPA on the Concentrations of Free and Sulfoconjugated Cathecholamines in Plasma, Cerebrospinal Fluid, Urine, and Central and Peripheral Nervous System Tissues of the Rat

The effects of subcutaneous injection of L-beta-3,4-dihydroxyphenylalamine (L-DOPA) on the concentrations of the catecholamines and catecholamine sulfates in the central and peripheral nervous systems of the rat were studied. The results showed that free 3,4-dihydroxyphenylethylamine (DA, dopamine) increased rapidly and markedly in the hypothalamus and striatum after L-DOPA but DA sulfate did not change. Increased concentrations of DA sulfate were detected in the CSF and in the plasma, where it reached a concentration of 130.8 +/- 12.8 ng/ml at 2 h, seven times the level of free DA (19.1 +/- 2.9 ng/ml). In the kidney the ratio of DA sulfate to free DA was reversed in favor of free DA. Urine samples of L-DOPA-treated rats showed a higher increase of free DA than DA sulfate, but free norepinephrine (NE) and NE sulfate remained unchanged. Concentrations of free DA and free NE in the adrenal glands of L-DOPA-treated rats showed no change. Adrenal DA sulfate and NE sulfate were not detectable in the control and L-DOPA-treated rats, suggesting that the adrenal glands lack the capacity to take up or store catecholamines and their sulfate counterparts from the plasma.