An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn

Immunohistochemical techniques have been used to characterise a number of interneuronal types found within the superficial layers of the rat dorsal horn. Substance P, methionine-enkephalin, neurotensin, avian pancreatic polypeptide, somatostatin and glutamic acid decarboxylase-like immunoreactivities were found within discrete populations of cell bodies within layers I to III of the spinal cord after colchicine pretreatment. Each immunoreactive cell type was distinct in terms of the distribution of terminals, perikaryal size and position within the dorsal horn. Cell bodies containing substance P were predominantly within layers I, II and III, while surgical isolation of the dorsal horn to remove terminals of extrinsic origin indicated that the terminals of the local substance P-containing interneurones were present throughout layers I and II₀. Enkephalin-immunoreactive neurones were most numerous within layer II, with less frequently observed larger enkephalin immunoreactive neurones in layer I. Neurotensin-containing neurones were found in a fairly narrow layer along the II/III border, with dendritic arborisations extending into both layers II and III. Neurotensin-immunoreactive terminals occurred in two bands, a dense band within layer II, and a less dense one within layers I and II₀. Somatostatin-like immunoreactivity was found in occasional large neurones within layer II_i. Avian pancreatic polypeptide immunoreactivity was localized to neurones within layers I, II and III, as was terminal immunoreactivity. Glutamic acid decarboxylase-containing neurones were large and were found predominantly within layers I, II ₀ and on the II/III border, where terminal immunoreactivity was also concentrated. Hemisection combined with dorsal root section indicated that the majority of these immunohistochemically identifiable neurones were interneurones.The interconnections and relations of these neurones to dorsal horn projection neurones are considered in the light of the reported analgesic properties of a number of these substances when applied directly to the spinal cord.     

Distribution, ontogeny and projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuron systems in the rat spinal cord: an immunohistochemical analysis

The distribution, ontogeny and fiber projections of cholecystokinin-8, vasoactive intestinal polypeptide and gamma-aminobutyrate-containing neuronal systems in the rat spinal cord were investigated by means of immunocytochemistry. Immunoreactive fibers to cholecystokinin-8, vasoactive intestinal polypeptide and glutamate decarboxylase (gamma-aminobutyrate-synthesizing enzyme, used as a marker of gamma-aminobutyrate) were widely distributed in the spinal cord, being particularly concentrated in the superficial dorsal horn, suggesting a close relationship to the pain transmission system. Cholecystokinin-8-containing neurons were mostly distributed in the dorsal laminae and glutamate decarboxylase-containing neurons were distributed in both the dorsal and ventral horns. Vasoactive intestinal polypeptide-containing neurons were detected in the lateral spinal nucleus and the lamina X. Cholecystokinin-8 and vasoactive intestinal polypeptide immunoreactive structures first appeared on gestational day 17-18. Although no substantial change in immunoreactive structures was observed during the fetal period, they increased markedly after birth. On the other hand, glutamate decarboxylase-positive structures appeared at gestational day 16 and those in the grey matter reached a maximum content at birth; both groups were present in adult animals. Transection of the upper cervical cord resulted in accumulations of cholecystokinin-8 and glutamate decarboxylase rostral to the lesion, revealing the presence of supraspinal projections of cholecystokinin-8 and glutamate decarboxylase to the spinal cord. The same experimental procedure demonstrated the existence of vasoactive intestinal polypeptide-mediating neuronal projections to the supraspinal level, as the accumulating fibers occurred in the area caudal to the lesion.     

Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens

An immunocytochemical analysis with 33 antisera was undertaken to investigate the localization of 25 different neurotransmitter-related antigens in the hypothalamic suprachiasmatic nucleus in the rat. To obtain estimates of relative densities of immunoreactive axons a stereological approach was used involving counting of intersections of immunoreactive axons with a superimposed semi-circle test grid. All neurotransmitter-related antigens found in perikarya within the suprachiasmatic nucleus, including those stained with antisera against bombesin, gastrin-releasing peptide, neurophysin, vasopressin, somatostatin, gamma-aminobutyrate, glutamate decarboxylase and vasoactive intestinal polypeptide were also found in axons within the nucleus. A greater number of these immunoreactive axons was found within the nucleus than in the adjacent anterior hypothalamus. The size of all immunoreactive axons in the suprachiasmatic nucleus was consistently small; immunoreactive axons were found ramifying widely in the nucleus, often ending with terminal boutons near perikarya immunoreactive for the same antigen. All neurotransmitter-related substances found in perikarya of the suprachiasmatic nucleus were also found in axons crossing over the midline to innervate the contralateral nucleus, providing an anatomical substrate for a high degree of communication between the paired nuclei. Axons immunoreactive for other putative transmitters including serotonin arising outside the nucleus were also found in high densities within the nucleus and crossing over the midline between the nuclei. Immunoreactivity for some transmitters was found in axons of similar densities within and outside the nucleus, including antisera against tyrosine hydroxylase; a small number of dopamine beta-hydroxylase and a few phenylethanolamine N-methyltransferase-immunoreactive axons were found in the SCN, suggesting that dopamine, norepinephrine and epinephrine may occur in a limited number of axons in the nucleus. Small numbers of axons immunoreactive with antisera raised against cholecystokinin, prolactin, substance P, thyrotropin-releasing hormone and choline acetyltransferase were found within the suprachiasmatic nucleus. Axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and neurotensin were rarely found within the suprachiasmatic nucleus; axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, cholecystokinin and tyrosine hydroxylase were found in both horizontal and coronal sections in the area between the left and right suprachiasmatic nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)     

Divergent effects of acute and chronic monosodium l-glutamate treatment on the anterior and posterior parts of the arcuate nucleus

Young albino rats were injected with either single or repeated doses of 0.1–6.0 mg/g body weight of monosodium l-glutamate between the ages of 2 and 40 days. The smallest, single effective dose was 0.25 mg/g body weight administered during the first week of life. The sensitivity to monosodium l-glutamate decreased with age. Monosodium l-glutamate caused nuclear pyknosis and edematous swelling of neurons in the anterior part of the arcuate nucleus which is located at the level of the ventromedial nucleus. These effects resulted in a reduction of neurons in this region. All of the doses used were ineffective in producing necrosis or cell death in the posterior part of the arcuate nucleus which is found at the level of the premamillary nuclei. The number of neurons in the arcuate nucleus of control animals was 46,500 with a neuron/glia ratio of 1.22. The anterior part of the arcuate nuclus had 55% of the neurons.Adult animals with an 80–90% loss of neurons in the anterior part of arcuate nucleus showed marked adiposity, hypoactivity, decreased body length, tail automutilation and reduced endocrine organ weight. The histological appearance of the ovaries, testes and pituitary glands was normal. The sexual behavior of these rats was normal, but the females were not fertile.The results of this study indicate that monosodium l-glutamate has a regional effect on the arcuate nucleus. These data also indicate that the majority of the neurons in the anterior arcuate region do not play a definitive role in the basal regulation of gonadotrophic hormones.     

The localization of acetylcholinesterase in the corpus striatum and substantia nigra of the rat following kainic acid lesion of the corpus striatum: a biochemical and histochemical study.

The distribution of acetylcholinesterase in the corpus striatum and substantia nigra was examined with the use of kainic acid lesions of the corpus striatum and pharmacohistochemical experiments. Histochemically identified acetylcholinesterase-containing neurons in the striatum were among those which were destroyed by kainic acid. Complementary biochemical studies demonstrated that approximately 50% of the total acetylcholinesterase activity in the striatum was localized in these acetylcholinesterase-containing neurons. Intrastriatal injections of kainic acid produced a substantial decrease in the activity of the glutamic acid decar?ylase in the substantia nigra, thus demonstrating that neurons contributing to the striato- and/or pallidonigral pathways had been lesioned. However, nigral acetylcholinesterase activity was not significantly reduced by the striatal kainic acid injections. Furthermore, stereotaxic injections of colchicine along the course of the striatonigral projection failed to produce an accumulation of acetylcholinesterase in these fibers proximal to the injection. In contrast, injections of colchicine into the nigrostriatal projection led to a proximal accumulation of acetylcholinesterase in the fibers of this system, thus confirming the presence of acetylcholinesterase in the ascending dopaminergic neurons.It is concluded that the striato- and pallidonigral projections in the rat do not contain significant levels of acetylcholinesterase. Furthermore, acetylcholinesterase-containing neurons in the striatum appear to be interneurons rather than the source of striatal efferents. It is suggested that some of these acetylcholinesterase-containing neurons may be striatal cholinergic interneurons.     

Vasoactive intestinal polypeptide afferents to the bed nucleus of the stria terminalis in the rat: an immunohistochemical and biochemical study

Vasoactive intestinal polypeptide (VIP) has a markedly heterogeneous distribution in the rat bed nucleus of the stria terminalis. The dorsal bed nucleus contains the highest concentration of VIP in the rat brain, with the exception of the suprachiasmatic nucleus, 4-fold higher than the VIP concentration in the frontal cortex. These biochemical findings agree well with the immunohistochemical analysis of this area. The bed nucleus is also a heterogeneous nucleus with respect to the afferent VIP pathways which innervate it. A combination of immunohistochemical and biochemical techniques was used to examine VIP innervation of the bed nucleus after knife cuts designed to interrupt ascending brainstem, stria terminalis and ventral amygdalofugal inputs to the bed nucleus. The results obtained suggest that (1) ascending pathways arising in the mesencephalon at the level of the dorsal raphe nucleus send VIP fibers to the dorsal but not the ventral bed nucleus, (2) afferent VIP fibers which travel to the bed nucleus via the stria terminalis contribute a diffuse VIP innervation to both the dorsal and ventral bed nucleus and (3) a newly described ventral amygdalofugal VIP pathway to the bed nucleus contributes a major input to the dorsal, but not to the ventral bed nucleus. These three pathways probably account for the entire extrinsic VIP input to the bed nucleus. The finding that the bed nucleus is heterogeneous both with respect to VIP content and afferent VIP inputs serves to clarify previous, apparently discrepant, reports that both the stria terminalis and ascending pathways constitute the major VIP input to the bed nucleus of the stria terminalis.     

Overall distribution of vasoactive intestinal polypeptide-containing nerves on the wall of cerebral arteries: an immunohistochemical study using whole-mounts

The overall distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity on the wall of the cerebral arteries, including its 3-dimensional profile, was investigated by means of the indirect immunofluorescence method using flat-mounts. VIP-immunoreactive fibers run spirally on the wall of the cerebral arteries. On the wall of the large arteries, such as the vertebral artery, basilar artery, internal carotid artery, within and/or without the circle of Willis, posterior and anterior communicating arteries, proximal parts of anterior, mid and posterior cerebral arteries, these fibers are richly distributed and show a dense grid-like appearance. The highest density was identified on the wall of the anterior cerebral artery, internal carotid artery and anterior communicating artery, while the lowest density was on the posterior communicating artery. On the other hand, on the walls of the branches of these arteries or along distal parts of the anterior, mid and posterior cerebral arteries, the number of VIP-immunoreactive fibers decreased markedly.     

Types of nerves in the enteric nervous system

The enteric nervous system is one of the three divisions of the autonomic nervous system, the others being the sympathetic and parasympathetic. In contrast to the other divisions, it can perform many functions independently of the central nervous system. It consists of ganglionated plexuses, their connections with each other, and nerve fibres which arise from the plexuses and supply the muscle, blood vessels and mucosa of the gastrointestinal tract. The enteric nervous system contains a large number of neurons, approximately 10⁷ to 10⁸. About ten or more distinct types of enteric neurons have been distinguished on electrical, pharmacological, histochemical, biochemical and ultrastructural grounds as well as on the basis of their modes of action. Both excitatory and inhibitory nerves supply the muscle and there are inhibitory and excitatory interneurons within the enteric plexuses. There are also enteric nerves which supply intestinal glands and blood vessels, but these receive less emphasis in this commentary.Correlations between groups of neurons defined on different criteria are poor and in many cases the physiological roles of the nerves are not known. The functions of noradrenergic nerves which are of extrinsic origin are reasonably well understood, but cholinergic nerves in the intestine are the only intrinsic nerves for which both the transmitter and to some extent the functions are known. In the case of non-cholinergic, non-noradrenergic enteric inhibitory nerves, the functions are understood but the transmitter is yet to be determined, both adenosine 5′-triphosphate and vasoactive intestinal polypeptide having been proposed. Other nerves have been defined pharmacologically (non-cholinergic excitatory nerves to neurons and muscle, intrinsic inhibitory inputs to neurons, and enteric, non-cholinergic vasodilator nerves) and histochemically (intrinsic amine-handling neurons and separate neurons containing peptides: substance P, somatostatin, enkephalins, vasoactive intestinal polypeptide, gastrin cholecystokinin tetrapeptide, bombesin, neurotensin and probably other peptides). Little is known of the functions of these nerves, although a number of proposals which have been made are discussed.     

Enhancement of photic shifts with the 5-HT1A mixed agonist/antagonist NAN-190: intra-suprachiasmatic nucleus pathway

Chronic desynchronization between the mammalian circadian pacemaker and its external environment, such as that observed from shift work or jet lag, can lead to various long-term health consequences. The circadian clock can be reset by exposure to light, although the magnitude of such adjustments is modest. 5-HT modulates the effects of light, and 5-HT_1A mixed agonist/antagonists, such as NAN-190, have been found to potentiate the phase resetting ability of light. The mechanism for this potentiation has yet to be uncovered, although it has been proposed that these drugs inhibit raphe output while simultaneously blocking post-synaptic 5-HT_1A receptors. The current study takes advantage of the heterogeneous network organization of the circadian clock to identify where in the circadian system NAN-190 exerts its influence. Retinorecipient cells in the ventrolateral suprachiasmatic nucleus (SCN) are activated by glutamate and release either gastrin-releasing peptide (GRP) or vasoactive intestinal polypeptide. Application of the glutamate agonist N-methyl-d-aspartic acid (NMDA) or either of these neuropeptides to the SCN mimics the effects of light. We hypothesized that NAN-190 would modify responses to treatments that activate the circadian system upstream, but not downstream, of where NAN-190 is acting. Hamsters were pretreated with NAN-190 or vehicle, followed by one of the neurochemicals 45 min later, during the early- and/or late-subjective night. NAN-190 potentiated NMDA-induced phase advances and delays as well as GRP-induced advances, but attenuated GRP-induced delays. NAN-190 did not potentiate NMDA-induced Fos expression, however greater GRP-induced Fos expression was found within the dorsolateral region of the SCN. These data suggest that NAN-190 acts, in part, by modifying the responsiveness of retinorecipient cells in the circadian clock. An understanding of the neural events that underlie the potentiation of photic phase shifts by NAN-190 could guide the development of novel chronobiotics which could be used to treat a variety of sleep and circadian disorders.     

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya.The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus.Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar?ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.     

The distribution and ultrastructure of VIP-immunoreactivity in the central nucleus of the rat amygdala

Vasoactive intestinal polypeptide (VIP) neurons within the central nucleus of the rat amygdala were examined using light and electron microscopic immunocytochemical techniques. Vasoactive intestinal polypeptide-immunoreactive neurons were located in the ventral part and less frequently in the central part of the central nucleus. Vasoactive intestinal polypeptide positive terminals were distributed throughout the medial part of a cytoarchitectonically distinct central zone of the central nucleus. Three types of terminals formed synaptic contacts on VIP-immunoreactive neurons: type A containing round vesicles, type B containing many pleomorphic vesicles and type C containing fewer pleomorphic vesicles. All VIP-immunoreactive boutons observed were of type A variety, and made asymmetrical and symmetrical synaptic contacts on both VIP-immunoreactive and nonreactive neurons within the central nucleus.     

Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat: distribution, possible origins and functional implications

The distribution of nerves containing vasoactive intestinal polypeptide(VIP)-immunoreactive material was examined in the cephalic arteries and cranial nerves of cats using an indirect immunofluorescence procedure on whole mounts. Perivascular VIP-immunoreactive nerves were widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-immunoreactive nerves were most abundant in the circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brainstem and ventral areas of the cerebral cortex. Nerves containing VIP-immunoreactive material were absent from distal portions of arteries supplying the posterior brainstem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-immunoreactive nerves had extracerebral origins probably from VIP-immunoreactive perikarya within microganglia in the cavernous plexus and external rete. Extracerebral perivascular VIP-immunoreactive nerves probably arose from VIP-immunoreactive perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. Therefore, it seems likely that each major segment of the cephalic circulation is supplied by local VIP-immunoreactive neurons. If the VIP-immunoreactive nerves cause vasodilation, they are well placed to allow redistribution of arterial blood flow within the head. During heat stress, neurogenic vasodilation of the appropriate beds would permit efficient cooling of cerebral blood, particularly that supplying the rostral brainstem and surrounding areas of the cerebral cortex.     

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. III. Synaptic interactions and modulatory effects of neurotransmitter candidates

We have used intracellular recordings to study synaptic interactions between myenteric neurons grown in dissociated cell culture. Intracellular stimulation of individual myenteric neurons caused several types of synaptic effects in nearby neurons: fast excitatory synaptic potentials mediated by nicotinic acetylcholine receptors; slow, non-cholinergic synaptic potentials; dual transmission having both fast cholinergic and slow non-cholinergic components and inhibition of spontaneously occurring fast nicotinic synaptic potentials. Fast nicotinic synaptic potentials were elicited by about 40% of neurons tested and often occurred spontaneously. The fast synaptic potentials were similar to those that have been studied in other autonomic neurons with respect to their estimated reversal potential and their sensitivity to cholinergic antagonists. The amplitudes of the fast synaptic potentials declined if evoked at frequencies greater than 0.5 Hz. Potentiation of the fast synaptic potentials was observed following high-frequency stimulation of presynaptic neurons. Several transmitter candidates modulated fast cholinergic transmission. Substance P and vasoactive intestinal peptide promoted nicotinic transmission by causing increased amplitudes of evoked and spontaneous fast synaptic potentials and an increased frequency of spontaneous synaptic potentials. gamma-Aminobutyrate and [Met]enkephalin both caused decreased amplitudes and frequency of nicotinic synaptic potentials. Serotonin depressed synaptic potentials in some neurons while enhancing them or having no effect in others. Slow, non-cholinergic, synaptic potentials were elicited by about 10% of neurons tested. These synaptic effects lasted 15-300s, caused depolarizations of 3-15 mv and were accompanied by increased neuronal input resistance. The transmitter(s) causing these slow synaptic potentials has not yet been identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kainic acid induced seizures: Neurochemical and histopathological changes

Behavioural, histopathological and neurochemical changes induced by systemic injection of kainic acid (10mg/kg, s.c.) were investigated in rats. The most pronounced behavioural changes were strong immobility (“catatonia”), increased incidence of “wet dog shakes”, and long-lasting generalized tonic-clonic convulsions. The behavioural symptoms were fast in their onset and lasted for several hours. Two distinct phases of histopathological and neurochemical changes were observed. (1) Early partially reversible changes were seen up to 3 h after kainic acid injection. They consisted of shrinkage and pycnosis of neuronal perikarya together with swelling of dendrites and axon terminals. These changes were accompanied by generalized signs of edema throughout the whole brain. Neurochemically there was a marked decrease in noradrenaline levels (up to 70%) and an increase in levels of 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid (up to 200%) in all analysed brain regions, suggesting a strongly increased firing rate of aminergic neurones during the period of generalized seizures. These histological and neurochemical changes were found in all the brain regions examined; they were greatly reduced or only sporadically seen after 1–3 days, when the animals had recovered from the seizures. (2) Late irreversible changes developed 24 h and later following kainic acid injection. They consisted of incomplete tissue necrosis with loss of nerve cells and oligodendrocytes, demyelination, astroglial scar formation, small perivenous hemorrhages and extensive vascular sprouting. The changes were restricted to the pyriform cortex, amygdala, hippocampus (most pronounced in the CA1 sector), gyrus olfactorius lateralis, bulbus olfactorius and tuberculum olfactorium. Neurochemically, a selective decrease was seen in choline acetyltransferase activity (40%) of the amygdala/pyriform cortex area, and of glutamate decar☐ylase activity in the dorsal hippocampus (45%) and amygdala/pyriform cortex (55%). No such changes were found in the frontal cortex and the striatum/pallidum. Since at these later time periods the widespread early changes in monoamine metabolism were mostly normalized, loss of acetylcholine and γ-aminobutyric acid neurons in the affected brain regions represented a selective neurochemical change typical for this stage of kainic acid action.

The observed neurochemical and histopathological changes may be directly related to tne excitotoxic and convulsive properties of kainic acid. However, brain edema resulting in herniation damage of the basal portions of the brain in addition to disturbances of microcirculation and anoxic-ischemic brain damage appear to be additional factors important in the pathogenesis of the late irreversible changes.     

Subcellular fractionation of cat submandibular gland: Comparative studies on the distribution of acetylcholine and vasoactive intestinal polypeptide (VIP)

In previous studies, evidence has been obtained for the presence of vasoactive intestinal polypeptide (VIP) in presumptive cholinergic neurons innervating the exocrine glands in the cat. In the present study, an attempt was made to define the storage sites of these two compounds in the cat submandibular gland using subcellular fractionation techniques. Particulate VIP was preferentially found in dense fractions (0.78–0.97 M sucrose) of a density gradient. Particle-bound acetylcholine showed a bimodal distribution in the gradient with relative enrichment in a dense fraction (0.87 M sucrose) but mainly in lighter fractions (0.28–0.44 M sucrose). More than 50% of all acetylcholine was recovered in a soluble form. For comparison, noradrenaline was also analysed and found in paniculate form in fractions ranging between 0.44–0.78 M sucrose. The electron microscopic analysis revealed presence of i.a. small clear vesicles in the lighter fractions, whereas the fractions rich in VIP contained i.a. many larger vesicles, sometimes with an electron-dense core. In agreement with earlier ultrastructural immunocytochemical studies on intact tissue, the present results support the view that VIP is present in large dense-cored vesicles. Most of the particulate acetylcholine seems to be localized in small clear vesicles, although a small proportion could be associated with large vesicles. Whether acetylcholine and VIP coexist in such large vesicles or whether separate populations of large vesicles exist, remains to be elucidated.     

Immunohistochemical studies of the enteric nervous system in tissue culture and in situ: localization of vascoactive intestinal polypeptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut

Immunofluorescence methods have been used to determine the detailed distribution of vasoactive intestinal polypeptide (VIP), substance-P and enkephalin nerve fibres in fixed cryostat sections from guinea-pig duodenum, jejunum, ileum, caecum at the site of the taenia coli, and proximal and distal colon. A novel method is used involving immunostaining of tissue culture preparations of both myenteric and submucous plexuses. These preparations allow each plexus to be studied in isolation from all axonal input for the first time, since they provide unequivocal extrinsic denervation together with severance of any intrinsic connections between the plexuses. In tissue sections the most prominent sites of VIP and substance-P immunoreactive fibres are the ganglia of the myenteric and submucous plexuses, the circular muscle layer and the longitudinal muscle of the taenia coli. In addition, VIP is prominent in the lamina propria of the submucosa except in the caecum. Enkephalin-immunopositive fibres are restricted to the ganglia of the myenteric plexus, the circular muscle layer and the longitudinal layer of the taenia coli. The culture preparations reveal that intrinsic ‘VIP neurons’ are common in the submucous plexus of the caecum and colon. They are also present, but in much lower numbers, in the myenteric plexus of the small intestine and colon but are not found in the myenteric plexus of the caecum. Intrinsic ‘substance-P neurons’ are present in the myenteric plexus from the small intestine, caecum and colon as well as in the submucous plexus of the colon; intrinsic ‘substance-P neurons’ are not found in the submucous plexus of the caecum. ‘Enkephalin neurons’ are numerous in the myenteric plexus of the small intestine, caecum and colon but are absent from the submucous plexus. Immunoreactivity is compared in the normal and denervated caecum by both the histochemical method and by radioimmunoassay of tissue extracts. In conjunction with the studies on tissue cultures, the results provide evidence for intrinsic reciprocal connections between the myenteric and submucous plexus of the caecum by neurons containing VIP and substance-P.An extensive comparison of these results with data from functional studies shows that the distribution of VIP, substance-P and enkephalin fibres in the gut is broadly in agreement with present knowledge of the action of these peptides on gut tissue, if it is assumed that they function as neurotransmitters or neuromodulators. In some instances, however, peptide-containing fibres and pathways are found which do not correlate with present knowledge obtained from functional studies. These observations provide new clues to the role of peptide neurons in gut function.     

Synaptic terminals in the dorsal lateral geniculate nucleus from neurons of the thalamic reticular nucleus: A light and electron microscope autoradiographic study

(³H)-proline was injected in the caudodorsal part (visual segment) of the thalamic reticular nucleus to study its projection to the dorsal lateral geniculate nucleus. This was done by autoradiographic tracing of anterograde axonal transport of tritium at the light- and electron microscopic level. The results of the light-microscope autoradiography show that connections of the thalamic reticular nucleus are distributed along lines of projections in the dorsal lateral geniculate nucleus, indicating a retinotopic arrangement of this projection. The results of the electron microscope autoradiography provide direct evidence that axons of cells in the thalamic reticular nucleus terminate in the dorsal lateral geniculate nucleus as synaptic boutons that contain flattened synaptic vesicles, dark mitochondria and establish symmetrical synapses with perikarya and with proximal, intermediate and distal dendrites. They do not take part in intraglomerular synapses (as boutons with pleomorphic synaptic vesicles do) and are not postsynaptic to other vesicle-containing boutons in the dorsal lateral geniculate nucleus.The present results, taken in conjunction with physiological studies that have shown postsynaptic inhibitory effects of the thalamic reticular nucleus on dorsal lateral geniculate nucleus relay cells in the rat, establish a correlation of an inhibitory synapse with the presence of flattened synaptic vesicles in the corresponding synaptic boutons. Also, the observation that thalamic reticular nucleus terminals in the dorsal lateral geniculate nucleus avoid forming synapses with boutons containing pleomorphic vesicles, believed to be synaptic processes of interneurons, is indicative that the inhibitory effects are exerted monosynaptically on geniculate relay cells.     

Distribution of neuropeptides in the limbic system of the rat: the bed nucleus of the stria terminalis, septum and preoptic area

The distribution of the neuropeptides vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin has been mapped immunocytochemically in the bed nucleus of the stria terminalis, one of the major sites of termination for efferent projections from the amygdala. Immunoreactive fibres and terminals were distributed more or less topographically and largely in accordance with the previously described localization of peptide-containing cell bodies in the amygdala and the amygdaloid projection fields in the bed nucleus as described by neuroanatomical techniques. Thus, vasoactive intestinal polypeptide, which was found in some of the lateral amygdaloid nuclei, had a substantial projection to the lateral bed nucleus. The lateral bed nucleus also contained cholecystokinin-octapeptide, substance P, neurotensin and methionine-enkephalin immunoreactivity which probably derived from the central amygdaloid nucleus, whilst cholecystokinin-octapeptide, and especially substance P-containing fibres, were found in the medial bed nucleus and probably arise from cells in the medial amygdala. Reciprocal amygdalopetal projections were suggested by the presence of substance P- and somatostatin-containing cell bodies in the mediodorsal bed nucleus and vasoactive intestinal polypeptide cells in the lateral bed nucleus, but somatostatin otherwise had a widespread distribution. Numerous local peptidergic connections were also noted both within the bed nucleus and between it and adjacent structures, especially the preoptic area, hypothalamus and the basal ganglia. These data provide further evidence that neuropeptides play a major role in the connectivity of the limbic system and show that the bed nucleus of the stria terminalis is an important relay station, particularly between amygdaloid efferents and other forebrain areas.     

Electrophysiology of the suprachiasmatic circadian clock

In mammals, an internal timekeeping mechanism located in the suprachiasmatic nuclei (SCN) orchestrates a diverse array of neuroendocrine and physiological parameters to anticipate the cyclical environmental fluctuations that occur every solar day. Electrophysiological recording techniques have proved invaluable in shaping our understanding of how this endogenous clock becomes synchronized to salient environmental cues and appropriately coordinates the timing of a multitude of physiological rhythms in other areas of the brain and body. In this review we discuss the pioneering studies that have shaped our understanding of how this biological pacemaker functions, from input to output. Further, we highlight insights from new studies indicating that, more than just reflecting its oscillatory output, electrical activity within individual clock cells is a vital part of SCN clockwork itself.