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Fine networks of phenylethanolamine N-methyltransferase (PNMT)-immunoreactive fibers are found in the hypothalamic paraventricular nucleus--mainly in the anterior, dorsal and dorso-medial parvicellular subdivisions, the lateral hypothalamus (dorsal, lateral and ventral to the fornix) and in the central amygdaloid nucleus. Coronal hemisections of the brainstem through the rostral level of the medulla oblongata show that most hypothalamic and amygdaloid PNMT fibers arise from the medullary adrenergic cell groups. Fourteen, but not 10 days after total hemisections, PNMT fibers disappeared almost completely from the hypothalamus and amygdala, ipsilateral to the knife cuts. A small decrease was also observed in the ventral, lateral hypothalamus on the contralateral side. Partial depletion of PNMT-immunoreactivity in the hypothalamus and the amygdala after medial or lateral brainstem hemisections indicates that ascending PNMT-immunoreactive fibers pass through mainly the lateral portion of the medulla, but some fibers also in its medial portion. Midsagittal transection of the diencephalon slightly reduced PNMT immunostaining in the paraventricular nucleus and the lateral hypothalamus bilaterally. The results show that the ascending PNMT system essentially is ipsilateral, but probably with a small crossing-over component, both at the diencephalic and lower brainstem level.

Using indirect immunofluorescence techniques combined with retrogradely transported fluorescent dyes, cholecystokinin (CCK)-like and substance P immunoreactive cell bodies in the periaqueductal central grey of the rat brain were studied. Data from both adjacent sections and elution-restaining techniques indicated that some of these central grey cells contain both a CCK-like peptide and substance P. Injection of the fluorescent dye, Fast Blue, into the cervical spinal cord indicated that this CCK-substance P cell group is a descending system. These findings provide evidence for two peptides in a single neuron, and the possible functional significance of such an occurrence is discussed.

Substance P is widely distributed in the nervous system. In brain and spinal cord it may act as a transmitter, for example at the central branches of primary sensory neurons. It may also be released from the sensory nerve endings and is thought to be involved in antidromic vasodilatation and in synaptic transmission in autonomic ganglia. In some central neurons substance P is stored together with 5-hydroxytryptamine and thyrotropin-releasing hormone. These neurons project to the ventral horn of the spinal cord, amongst other places. In another system substance P coexists with a cholecystokinin-like peptide. These neurons are localized in the periaqueductal central grey matter and also project to the spinal cord. Finally, injection of a substance P antagonist into the ventral mesencephalon causes marked morphological changes in neurons that contain dopamine, substance P and gamma-aminobutyric acid (GABA).

High amplitude spiking representative of seizures, accompanied by an unusual motor behavior pattern of rearing and forelimbic clonus resembling "boxing," was elicited by microinjection of the cholinergic agonist, carbachol, 4 micrograms, into the medial prefrontal cortex of the rat. A rating scale devised to score the behavior revealed a motor pattern elicited by carbachol from the medial anterior cortex which was similar to that described by Racine for electrical stimulation of the amygdala. Topographical analysis of the areas surrounding the medial anterior cortex region revealed that the motor manifestations of seizures were elicited over a wide region of the anterior cortex, with scores significantly lower at carbachol microinjection sites greater than 1 mm rostral, 2 and 3 mm caudal, and 2 mm lateral to the standard medial prefrontal cortex site. Unilateral microinjection of carbachol yielded motor seizures primarily from the contralateral forepaw, suggesting involvement of a crossed pathway. Retrograde tracing with fast blue dye, combined with immunostaining for choline acetyltransferase and NADPH-diaphorase, found that the cholinergic neurons innervating the standard microinjection site were the dorsolateral tegmental cells, as previously reported, which have been shown to also contain substance P and corticotropin releasing factor. In addition, cholinergic neurons of the nucleus basalis of Meynert region were found to innervate the standard microinjection site. These findings implicate cholinergic innervation of the rostral cortex in classical limbic seizures.

Sulfated cholecystokinin octapeptide (CCK) potentiates dopamine-induced hyperlocomotion in the nucleus accumbens of the rat. Immunocytochemical evidence has shown a topographical distribution of terminals containing both CCK and dopamine (DA), within the medial posterior nucleus accumbens. Seven sites within the nucleus accumbens were cannulated and tested for the ability of CCK to enhance the behavioral effects of DA. Close agreement was found between the anatomical sites of CCK-DA coexistence, and the anatomical sites at which CCK potentiated DA-induced hyperlocomotion. Behaviorally inactive sites were found primarily in the anterior nucleus accumbens, where DA-containing terminals do not contain CCK.

The possible sources of neurotensin-like immunoreactive axons in the median eminence were studied after several experimental surgical approaches including unilateral lateral retrochiasmatic area transection, midsagittal knife cut through the median eminence, complete surgical isolation of the medial basal hypothalamus and bilateral paraventricular nucleus lesions. Both immunohistochemical and radioimmunoassay data demonstrate that neurotensin-containing neuronal somata located in the hypothalamic arcuate nuclei represent the main source of neurotensin occurring in the external zone of the median eminence of the rat: neither the complete isolation of the medial basal hypothalamus nor the transection of the major neuronal input channel to the median eminence in the lateral retrochiasmatic area altered neurotensin-like immunoreactivity in the median eminence; bilateral lesioning of the paraventricular nucleus resulted in insignificant changes of neurotensin level in the median eminence; and two days after lesioning the median eminence an increased amount of retrogradely accumulated neurotensin-like immunoreactivity was found in several perikarya of the arcuate nuclei due to the blockage of axonal transport in the transected fibers. Retrograde accumulation of neurotensin-like material in other cells scattered in the anterior hypothalamus (in the paraventricular, periventricular and anterior hypothalamic nuclei) indicates that in addition to the arcuate neurons these neurons may also participate in the neurotensin innervation of the median eminence.

The effects of kainic acid lesions and chronic haloperidol treatment on rat striatal dopaminergic presynaptic receptors were studied. Following the gamma-butyrolactone-induced inhibition of dopaminergic impulse flow, and after dopa decarboxylase inhibition, dopa accumulation and its reversal by dopamine agonists was measured in vivo. 3H-apomorphine (a dopamine receptor ligand with purported presynaptic specificity) was used for in vitro binding experiments. Presynaptic dopamine receptors, as assessed by both methods, were unaffected by intrastriatal kainic acid injection 5-6 days before sacrifice. Seven days after termination of chronic haloperidol treatment (28 days, 0.5 mg/kg/day s.c.) both an increased apomorphine response using the dopa accumulation method and an increase in 3H-apomorphine binding were observed, indicating the development of presynaptic dopamine receptor supersensitivity.

Extracellular recordings of single unit activity were made in the substantia nigra (SN) of chloral hydrate-anaesthetized rats. Dopaminergic neurones of the pars compacta (SNC) were stimulated by (-)-nicotine bitartrate (1.0 mg kg-1) given subcutaneously (s.c.). This action was prevented by the secondary amine mecamylamine HCl (2.0 mg kg-1 i.v.) but not by a ganglion-blocking dose of the bisquaternary compound chlorisondamine Cl (0.1 mg kg-1 i.v.). Mecamylamine reduced the spontaneous activity of dopaminergic neurones. Nicotine, when administered intravenously (2-128 micrograms kg-1 cumulative dose), also stimulated dopamine cells and this action was dose-related. Nicotine, administered intravenously, (2-128 micrograms kg-1 cumulative dose) markedly excited non-dopamine cells in the pars reticulata (SNR) in a dose-related manner. In rats pretreated with chlorisondamine (0.1 mg kg-1 i.v.), nicotine induced a small excitatory or depressant action, but the marked excitation was not seen. Mecamylamine (2 mg kg-1 i.v.) completely prevented the actions of nicotine. The results are consistent with a direct excitatory action of nicotine on dopaminergic neurones of the substantia nigra pars compacta. The pronounced excitatory action of systemically administered nicotine on non-dopamine cells of the pars reticulata appears to be of peripheral origin.

The effects of increased extracellular calcium levels on the responses of the motor nerve terminal to agents that increased intracellular levels of cyclic AMP was studied on the in vivo cat soleus muscle preparation. The neural and muscle responses to intra-arterially administered NaF, an activator of adenylate cyclase, and dbcAMP progressively increased as blood calcium levels rose. These results provide additional support for the hypothesis of an interaction between calcium and cyclic AMP in motor nerve endings.

Cholecystokinin (CCK)-like peptides when administered intravenously produce 2 distinct actions on the single-unit activity of mesencephalic dopamine (DA) neurons in the rat: an excitatory action and a potentiation of the inhibitory effects of DA agonists. The ability of several CCK fragments that have been shown to bind selectively to the peripheral and/or the central CCK-binding sites were examined for their ability to induce either excitation or a potentiation of DA. Only sulfated CCK-8 was able to induce excitation of mesencephalic DA neurons, but both sulfated and unsulfated CCK-8, as well as CCK-4, potentiated the inhibitory effects of the DA agonist apomorphine (APO). CCK-3 failed to potentiate APO-induced inhibition. Both of these effects appeared to be confined to cell bodies in regions of the ventral tegmental area and substantia nigra, zona compacta that have been reported to contain both DA and CCK. Thus, CCK-like peptides that have been shown to bind to the high-affinity CCK binding site in brain potentiated the effects of DA. In contrast, the ability of CCK-like peptides to induce neuronal excitation corresponds with their affinity for the peripheral-type CCK binding site.

Previous studies have demonstrated a neuronal system containing an immunoreactive ranatensin-like peptide (irRT) in rat brain. The results of the present study describe the ascending projections of a prominent irRT cell group in the pons. A major pontine irRT fiber system was shown to innervate the hippocampus but not the mammillary body.

The distribution of low-affinity sodium-sensitive binding sites of [3H]mazindol were studied in rat hypothalamic nuclei. Using microdissection methods, it was demonstrated that the highest level of [3H]mazindol binding is localized to the paraventricular nucleus (PVN) and the lowest binding is observed in the lateral hypothalamus. Following food deprivation, a significant decrease in [3H]mazindol binding in the PVN and ventromedial hypothalamus (VHM) were observed. Refeeding food-deprived rats resulted in restoration of the level of binding in the PVN, and this was correlated with changes in blood glucose levels. Thus, changes in the binding of [3H]mazindol in the PVN may reflect local changes in glucose levels. In related studies, the involvement of the PVN in the regulation of food deprivation or 2-deoxyglucose (2-DG)-induced food intake was studied. Application of amphetamine (20 micrograms) into the PVN had no effect on food deprivation induced feeding, but significantly inhibited 2-DG induced (glucoprivic) feeding. The PVN may play an important role in the glucostatic regulation of feeding and in mediating the anorectic action of amphetamine and related anorectic drugs on glucoprivic feeding.

Cholecystokinin (CCK) and dopamine (DA) co-exist in ventral tegmental neurons which project via the mesencephalic pathway to the nucleus accumbens of the rat. CCK and DA are located in separate neurons in the substantia nigra which projects via the nigrostriatal pathway to the caudate nucleus in the rat. The functional significance of this peptide-amine co-localization was investigated using behavioral and neurophysiological techniques. CCK injected directly into the nucleus accumbens potentiated apomorphine-induced stereotypy and dopamine-induced hyperlocomotion. CCK injected directly into the caudate nucleus had no effect on apomorphine-induced stereotypy or dopamine-induced hyperlocomotion CCK injected alone into either site did not induce stereotypy or hyperlocomotion. The dose-response curve to apomorphine induction of stereotypy was shifted to the left by CCK, indicating increased sensitivity to the dopaminergic agonist. Neurophysiological analysis of the firing rate of ventral tegmental neurons demonstrated that CCK produced a left-shift in the dose-response curve of apomorphine on inhibition of neuronal firing. These data suggest that CCK acts as a modulator of dopamine, increasing neuronal responses to dopaminergic agonists. The potentiation of dopamine by CCK may be specific to the mesolimbic neurons, where CCK and DA co-exist in the rat.

In response to stress, adrenocorticotropic hormone (ACTH) is released by corticotrophs in the anterior pituitary under the control of several central and peripheral factors including corticotropin-releasing factor (CRF), which was recently isolated from the brain and sequenced. Immunocytochemical studies have shown that most of the CRF-containing cell bodies that project to the median eminence are present in the hypothalamic paraventricular nucleus (PVN). A dense PNMT(phenylethanolamine-N-methyltransferase)-containing fibre network was also observed in the same region--PNMT is the final enzyme in the biosynthesis of adrenaline and has been demonstrated in the brain. In the present study we found an association of adrenergic nerve fibres and CRF neurones by immunohistochemistry using antisera to PNMT and CRF. To examine the functional significance of the adrenergic projection to the PVN, we blocked the synthesis of adrenaline using a specific inhibitor of PNMT. The depletion of adrenaline resulted in an increase in CRF immunoreactivity. The present results suggest that, as well as catecholamines which regulate ACTH release at the anterior pituitary level via a beta 2-adrenergic receptor mechanism, central catecholamines (mainly adrenaline) also affect ACTH release through their action on CRF cells. Peripheral catecholamines seem to have a direct stimulatory effect on the pituitary corticotroph cells, whereas the present findings suggest that central adrenaline-containing neurones have an inhibitory role in the physiological response to stress.

This research explored the possibility that cyclic nucleotides are part of the excitation-secretion sequence in mammalian motor nerve terminals. A series of reagents known to react with the enzymes that synthesize and degrade cyclic nucleotides or that are effectors of cyclic nucleotide actions were administered to in vivo cat soleus nerve-muscle preparations. The reagents were administered by rapid close intra-arterial injection while electrical activity in single motor axons and contractile activity of the muscle were monitored. NaF, an activator of adenylate cyclase, evoked bursts of action potentials in unstimulated axons and caused stimulus-bound repetitive activity in stimulated axons. It evoked vigorous asynchronous activity in the muscle and potentiated the force of muscle contraction. These effects are identical with those of cyclic N6-2'-O-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP). Prostaglandin E1 produced similar effects. Dithiobisnitrobenzoic acid and alloxan, inhibitors of adenylate cyclase, impaired neuromuscular transmission and prevented the effects of NaF, but they did not change the responses to dibutyryl cAMP. Theophylline, an inhibitor of phosphodiesterase, caused axons to respond repetitively to stimulation, but this activity had a different pattern from that produced by dibutyryl cAMP or NaF. Pretreatment with theophylline enhanced the responses to dibutyryl cAMP and NaF. Imidazole, an activator of phosphodiesterase, impaired neuromuscular transmission and prevented the effects of dibutyryl cAMP and NaF. Adenosine, an inhibitor of protein kinase, or verapamil, which inhibits calcium flux, impaired neuromuscular transmission and prevented the responses to dibutyryl cAMP, NaF and theophylline. These results are compatible with the hypothesis that cAMP is involved in the regulation of calcium flux and transmitter secretion in mammalian motor nerve terminals.

Pharmacological studies on the behavioral functions of sulfated cholecystokinin (CCK) in the gut and in the brain require potent, specific antagonists to CCK. Compounds identified as competitive antagonists at the peripheral receptors for CCK were tested for their ability to block the behavioral effects of CCK administered centrally and peripherally. Behavioral effects of CCK (8.8 X 10-10 mmol) administered centrally into the nucleus accumbens, i.e., potentiation of dopamine-induced hyperlocomotion in rats, were effectively blocked by pretreatment with proglumide (6 X 10(-5) mmol of nucleus accumbens), by benzotript (3 X 10(-5) mmol of nucleus accumbens) and by rabbit antiserum raised against CCK (0.2 microliter/nucleus accumbens), but not by CCK26-33 (1.7 X 10(-7) mmol) or unsulfated CCK26-33 (1.9 X 10(-6) mmol). The behavioral effects of peripherally administered CCK, i.e. reduced food consumption and reduced exploratory behaviors in mice, were blocked effectively by pretreatment with proglumide (0.3-0.9 mmol/kg), and by benzotript (0.03 mmol/kg), but not by CCK30-33 (0.003 mmol/kg). None of the compounds administered peripherally significantly affected food consumption or exploratory behaviors when given alone. Furthermore, none of the compounds significantly affected locomotion when administered alone into the nucleus accumbens, or significantly affected dopamine-induced hyperlocomotion when given into the nucleus accumbens before dopamine. Benzotript, proglumide and a CCK antibody appear to act as specific antagonists of the behavioral effects of CCK at both the peripheral gastrointestinal site and at the central nucleus accumbens site. Neither unsulfated CCK26-33 or CCK30-33 were effective as antagonists of peripheral or central behavioral effects of CCK. However, whereas benzotript and proglumide may be useful as pharmacologically specific antagonists, the high doses required suggest that more potent CCK antagonists are required for investigating the behavioral functions of endogenous CCK.

Interactions between neuronal responses mediated by dynorphin A1-8 and GABA were investigated in the substantia nigra zona reticulata. Extracellular recordings and microiontophoresis were performed using five-barrel microelectrodes in chloral hydrate-anesthetized male rats. When iontophoresed alone, dynorphin A1-Q significantly inhibited the firing of 22% of the neurons tested. The inhibition was rapid in onset and recovery and was dose-dependent. In another 22% of the cells, iontophoretic dynorphin produced an increase in the baseline firing rate which was slow in both onset and offset; the remaining 56% were unaffected by dynorphin. When GABA and dynorphin A1-8 were applied in conjunction, the inhibitory action of GABA was attenuated in 61% of the cells; whereas, when dynorphin and GABA were ejected simultaneously onto the cells that were inhibited by dynorphin A1-8, the respective inhibitory effects of dynorphin and GABA appeared to be additive. The kappa antagonist, MR-2266, failed to block the ability of dynorphin A1-8 to attenuate the action of GABA. In addition, the non-opiate peptide des-tyr-dynorphin A2-17, produced effects similar to that of dynorphin A1-8. The role of dynorphin in the basal ganglia and its interaction with the other major transmitter in the substantia nigra zona reticulata, GABA, is discussed.

The effects of verapamil on the force of contraction of soleus and gastrocnemius muscles of the cat were examined in vivo. The rapid intra-arterial administration of verapamil (100-800 micrograms/kg) produced a dose-dependent increase in the contractile force of soleus and gastrocnemius muscles stimulated supramaximally via their nerves. Verapamil also potentiated twitch tension in d-tubocurarine-treated, directly stimulated soleus preparations. This agent had no effect, however, on the contractile force of 14- to 16-day chronically denervated, directly stimulated soleus muscle. Verapamil (400 microgram/kg, i.a.) also produced a significant increase in muscle action potential duration while having no effect on amplitudes, rise times or resting membrane potential. It is concluded that verapamil acts directly on innervated skeletal muscle to increase muscle force and it is suggested that the probable site of action is at the muscle membrane.

Three groups of gamma-aminobutyric acid--containing neurons were found in the mammillary region of the posterior hypothalamus. The groups correspond to the tuberal, caudal, and postmammillary caudal magnocellular nuclei. Many cells in these nuclei were retrogradely labeled with fast blue after the injection of this fluorescent dye into the neocortex. Immunohistochemical experiments showed that these same neurons also contained the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase. These results provide morphological evidence for a gamma-aminobutyric acid pathway arising in magnocellular neurons of the posterior hypothalamus and innervating the neocortex.

The distribution of substance P and leucine enkephalin in mid- and fore-brain areas of the rat was studied using a radioimmunocytochemical method. The secondary antibody was labeled with 125I and the sections apposed to LKB Ultrofilm or emulsion-dipped. In alternate sections an extensive distribution of substance P and enkephalin immunoreactive material was seen in frontal, cingulate, retrosplenial, and entorhinal cortices. Substance P and enkephalin exhibited a remarkable overlap in many of these cortical areas as well as in the nucleus accumbens, caudate, portions of the hypothalamus, amygdala, thalamus and central gray. Differences in distribution were seen in the retrosplenial cortex, septum, ventromedial hypothalamus, hippocampus, the substantia nigra and the superior colliculus. The results provide a detailed immunohistochemical demonstration of the laminar patterns of substance P and enkephalin in the cortex of the rat. The results are discussed in terms of the interaction of substance P and enkephalin. The matches and mismatches of immunoreactive substance P and enkephalin and the locations of their receptors are also examined.

In a modification of the indirect immunohistochemical method 125I-labeled secondary antibodies were used to autoradiographically visualize enkephalin-like immunoreactivity. Electrolytic lesions of the bed nucleus of the stria terminalis (BNST) resulted in a decrease in enkephalin-like immunoreactivity in the ipsilateral habenula. This suggests an opiatergic pathway originating in the BNST and projecting to the habenula. In addition, the value of the radioimmunohistochemical technique is discussed.

Retrograde tracing with a fluorescent dye (Fast Blue) combined with immunohistochemistry was used to identify putative neurotransmitter(s) at the phrenic motor nucleus in the cat. Fast Blue was injected bilaterally into the diaphragm of five cats, where each phrenic nerve enters the muscle. Seven days later the animals were perfusion fixed and tissue sections from the fourth, fifth, and sixth cervical spinal cord segments were analyzed using a fluorescence microscope. Retrogradely labeled fluorescent phrenic motor neuron cell bodies appeared in all of the segments but primarily in sections from the fifth segment. The same or adjacent transverse sections were then used for the demonstration of the distribution of the neurotransmitters 5-hydroxytryptamine (5-HT), substance P, and thyrotropin-releasing hormone (TRH) in the area of the phrenic motor nucleus using the indirect immunofluorescence technique. The most conspicuous neurotransmitters found at the phrenic motor nucleus were 5-HT and substance P. We observed dense and diffuse fiber networks throughout the ventral horn which contains the phrenic motor nucleus. These fibers contained varicosities in close proximity to phrenic motor neurons. In addition to 5-HT- and substance P-containing nerve endings, some fibers containing TRH were also found in the area of the phrenic motor nucleus. These results are consistent with earlier physiological data suggesting that 5-HT, substance P, and TRH are important neurotransmitters and/or neuromodulators involved in central control of respiration.

The immunoperoxidase technique was used to study the effect of adrenalectomy on vasopressin (VP) immunoreactivity in the hypothalamic paraventricular nucleus of rat. In control animals, relatively few VP-immunostained parvocellular neurons were found in addition to a large population of magnocellular VP neurons. Seven to 14 days after bilateral adrenalectomy, VP immunostaining increased markedly in specific subdivisions of the paraventricular nucleus. In contrast to normal animals, VP immunoreactivity was localized in a large number of parvocellular neurons. Colchicine treatment, on the other hand, did not significantly increase the number of VP-immunostained parvocellular neurons found in control rats. These observations suggest that adrenalectomy increases the number of VP-positive neurons and appears to increase the intensity of VP immunoreactivity specifically in parvocellular neurons. VP parvocellular neurons are confined to those paraventricular nucleus subdivisions that are known to project to the external zone of the median eminence. Moreover, their distribution pattern is very similar, if not identical, to that of the corticotropin-releasing factor (CRF) immunoreactive cells. Parvocellular neurons on adjacent thin sections could be stained for both CRF and VP. Thus, adrenalectomy seems to increase VP staining in CRF immunoreactive parvocellular neurons, which innervate the external zone of the median eminence.

In this study, we investigated the effects of acute caffeine administration on the activity of midbrain dopamine neurons. Caffeine significantly depressed the firing rates of dopamine neurons in the ventral tegmental area (A10 group), but had no significant effect on the firing rates of dopamine neurons in the substantia nigra zona compacta (A9 group). The action of caffeine in A10 was completely blocked by pretreatment with the adenosine agonist L-phenyl-isopropyl-adenosine (L-PIA), confirming numerous lines of evidence that caffeine and other xanthines act as competitive antagonists at adenosine receptors. The dopamine antagonist haloperidol also antagonized the effects of caffeine. This finding is consistent with a mechanism of caffeine-induced depression of dopamine neuron activity involving dopamine release, similar to that observed during amphetamine administration. Finally, the benzodiazepine diazepam also antagonized the dopaminergic effects of caffeine. It appears that, in the rat, caffeine administration inhibits mesolimbic and mesocortical projecting dopamine neurons, but has no effect on dopamine neurons that project to the striatum.