GABA-immunoreactive neurons and terminals in the lateral cervical nucleus of the cat

Previous findings have indicated the presence of local circuit neurons in the lateral cervical nucleus (LCN). An immunohistochemical study with gamma-aminobutyric acid (GABA) antiserum was therefore performed both to investigate whether GABA-immunoreactive neurons are present in the LCN, and if so, to compare their characteristics with those previously assigned to probable internuncial neurons in the nucleus. The fine structure and synaptology of GABA-positive boutons in the LCN were also studied. Transversely cut sections from the upper cervical spinal cord of three cats were processed for GABA immunohistochemistry with the free-floating PAP technique. On light microscopic examination immunoreactive neurons were observed within the ventromedial half of the LCN. Their total number was estimated to be 42.5 +/- 11.7 in the entire LCN on one side of the cervical spinal cord, but this may have been an underestimation, as the penetration by the antisera was limited. The labeled neurons were small and had a relatively large nucleus and a low bouton covering ratio. In their number, localization, and ultrastructural appearance the GABA-positive neurons closely resembled the population of neurons previously suggested to be local circuit neurons. Immunoreactive bouton-sized puncta were scattered throughout the LCN. Ultrastructural examination showed labeled terminals with a mean sectional area of 0.85 micron 2 and a relatively high density of synaptic vesicles. The vast majority of GABA-positive terminals were in contact with dendrites and only a minority had synaptic contact with cell bodies. No axoaxonal synapses were observed. The GABA-positive boutons probably derive at least partly from the observed GABA-positive neurons, but there is also a possibility of extrinsic GABAergic input.     

GABA-immunoreactive neurons and terminals in the lateral cervical nucleus of the cynomolgus monkey

An antiserum against the inhibitory transmitter substance gamma-aminobutyric acid (GABA) was used to investigate the distribution of GABAergic nerve terminals and cell bodies in the lateral cervical nucleus (LCN) of the cynomolgus monkey. Light microscopic immunohistochemistry demonstrated GABA-immunoreactive puncta, suggestive of nerve terminals, scattered throughout the LCN. The terminal-like profiles are often present along the somata of unlabeled neurons, but most are located in the neuropil. GABA-immunoreactive neurons are present in the LCN, but constitute a very small number of the LCN neurons. Electron microscopy showed that the GABA-positive neurons are small with a relatively large nucleus. They are contacted by few somatic boutons. Numerous GABA-immunoreactive terminals containing densely packed round to oval synaptic vesicles were also found. Most GABA-positive terminals make synaptic contact with dendrites, but synapses with cell bodies are also present. Synaptic contacts between labeled and unlabeled terminals were not observed. Some GABA-positive terminals make contact with GABA-positive neurons. The present findings suggest that GABA is a major inhibitory transmitter substance in the LCN of the monkey. However, in comparison with other somatosensory relay nuclei, there are few GABA-immunoreactive neurons in the LCN. This may imply that the GABA-positive neurons branch extensively in the LCN or that an extrinsic source of GABAergic input exists.     

GABA-immunoreactive neurons in the rat cerebellum: a light and electron microscope study

An antibody raised against gamma - amino-butyric acid (GABA) coupled to bovine serum albumin with glutaraldehyde (Hodgson et al: J. Histochem. Cytochem. 33:229-239, '85) was used to localise immunocytochemically the presumptive GABAergic neuronal elements in the cerebellar cortex of the adult rat. employing the unlabelled antibody enzyme method with pre- and post-embedding immunocytochemical procedures, the following cellular structures were observed to be GABA-immunopositive in both the light and electron microscopes: the somata, dendrites, and axonal processes (including axon terminals) of stellate, basket, and Golgi neurons. In immunopositive neuronal somata and dendrites, the reaction product was found to be associated with all intracellular organelles and with the postsynaptic densities of synaptic junctions. Specific GABA-like immunoreactivity was also seen around outer mitochondrial membranes, microtubules, and neurofilaments, and coating synaptic vesicles in presynaptic axon terminals. In the pre-embedding procedure with dilutions of the antiserum between 1:1,000 and 1:2,000, the perikarya and dendrites of Purkinje cells were GABA-immunonegative, whereas at an antiserum dilution of 1:500 the somata of Purkinje cells were mildly GABA-immunoreactive. Purkinje cell axon terminals in the infra- and supraganglionic plexuses and in the deep cerebellar nuclei were always strongly immunopositive. Neuroglia were invariably GABA-immunonegative, as were the dendrites, axons (parallel fibres), and somata of granule cells. Mossy fibre and climbing fibre afferents were also immunonegative. The pattern of immunoreactivity obtained with this antiserum directed against the inhibitory neurotransmitter GABA was found to resemble closely the immunocytochemical distribution of GABA and of the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) as reported previously in other immunocytochemical investigations (Oertel et al. and Wu et al: Cytochemical Methods in Neuroanatomy. New York: A. R. Liss, '82; Seguela et al: Neuroscience 16:865-874, '85; Mugnaini and Oertel: GABA and Neuropeptides in the CNS. Handbook of Chemical Neuroanatomy, Vol. 4, Part I. Amsterdam: Elsevier, '85.     

An ultrastructural study of neurotensin-like immunoreactive terminals in the mediodorsal thalamic nucleus of the rat.

Neurotensin-like immunoreactive (NTir) axon terminals in the mediodorsal nucleus of the thalamus (MD) in the adult rat were demonstrated by electron microscopic immunohistochemistry. Most NTir terminals were large (greater than 2 microns in diameter) with round synaptic vesicles and asymmetrical synaptic contacts although smaller (less than 1.5 microns in diameter) axon terminals were also labeled. Both types of terminals were found in the medial and central parts of MD with the greatest density in the medial part. These NTir boutons have similar ultrastructural features as anterogradely labeled terminals from the piriform cortex and the preoptic area, which have previously been identified as sources of NTir axons in MD. A few NTir boutons were also found in the medial part of MD with pleomorphic vesicles and symmetrical synaptic contacts.     

Cholinergic neurons in the rat septal complex: ultrastructural characterization and synaptic relations with catecholaminergic terminals.

Physiological and pharmacological studies have suggested that catecholamines modulate cholinergic neurons in the medial septal and diagonal band nuclei (i.e., the septal complex). Thus, the ultrastructural morphology of neurons containing choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, and their relation to catecholaminergic terminals exhibiting immunoreactivity for the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) were examined in the rat septal complex. Dual immunoautoradiographic and peroxidase anti-peroxidase labeling methods were used to simultaneously localize antibodies raised in rabbits against TH and from rat-mouse hybridomas against ChAT in single sections. At least two types of perikarya with ChAT-immunoreactivity (ChAT-I) were observed. The first type were large (20-30 microns), elongated or round, and contained a small indented nucleus with an abundant cytoplasm and an occasional lamellar body. The second type was also either ovoid or round but was medium-sized (15-20 microns) and contained a larger indented nucleus and a smaller amount of cytoplasm than the first type. Both types of perikarya as well as dendrites with ChAT-I were surrounded by astrocytic processes apposed to most of their plasmalemmal surfaces. The distribution and types of terminal associations (i.e., asymmetric synapses, symmetric synapses and appositions which lacked a membrane specialization in the plane of section analyzed) with ChAT-labeled perikarya and dendrites were quantitatively evaluated. The majority (68% of 197) of the presynaptic terminals were unlabeled; the remaining terminals were immunoreactive for TH (25%) or ChAT (7%). All three types of terminals contacted primarily the shafts of small dendrites and more rarely ChAT-labeled perikarya and large dendrites. ChAT-labeled terminals: (1) formed associations with unlabeled perikarya and dendrites (31% of 176); (2) formed associations with perikarya and dendrites with ChAT-I (7%); (3) contacted the same unlabeled perikarya and dendrite as a TH-containing terminal (21%); (4) were in apposition to TH-labeled terminals (25%); or (5) were either in apposition to unlabeled or ChAT-labeled terminals or lacked associations with any processes. The majority of associations formed by the terminals with ChAT-I were on the shafts of small dendrites. Moreover, most of the associations formed were either symmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. These findings provide cellular substrates in the septal complex (1) for sparse synaptic input relative to astrocytic investment of cholinergic neurons and (2) for direct synaptic modulation of cholinergic and non-cholinergic neurons by catecholamines and/or acetylcholine. These findings have direct relevance to catecholaminergic-cholinergic interactions and to the neuropathological basis for Alzheimer's disease.     

Decrease of GABA-immunoreactive neurons in the amygdala after electrical kindling in the rat

The present study was designed to investigate the effects of electrical kindling in vivo on GABA immunoreactivity (GABA-IR) of the lateral and basolateral amygdaloid nuclei 2-6 months post-stimulation. Male Sprague-Dawley rats were implanted with bipolar electrodes in the basolateral nucleus and stimulated once per day until 3-5 stage 5 seizures were observed. Coronal sections containing the amygdala were processed for GABA-IR using the contralateral side of the brain. Results indicate that, in comparison to controls, fully kindled animals showed a significant decrease in total number of GABA-IR amygdala neurons. Decreases in GABA-positive punctate structures surrounding unlabeled pyramidal cells were also observed, but not quantified. The present data suggest that epileptogenesis of the amygdala is associated with a significant reduction of GABA-IR in the lateral and basolateral areas throughout the contralateral amygdaloid nucleus.     

Callosal terminals in the rat prefrontal cortex: Synaptic targets and association with GABA-immunoreactive structures

The callosal projections of the cerebral cortex play an important role in the functional integration of the two hemispheres, and the anatomy of these connections has been extensively studied in primary sensory and motor regions. In the present investigation, we examined the synaptic targets of callosal terminals in a limbic association area, the prefrontal cortex (PFC) in the rat. In addition, we examined the relationship of callosal afferents to GABA local circuit neurons within the PFC. Callosal terminals were labeled by either anterograde transport of Phaseolus vulgaris leucoagglutinin from superficial or deep layers or by anterograde degeneration following electrolytic lesion of the contralateral PFC. Callosal terminals in either the superficial or deep layers labeled by either method formed primarily asymmetric axo-spinous synapses (approximately 95%), while the remainder formed axo-dendritic synapses. Some of the dendrites postsynaptic to callosal terminals exhibited a morphology characteristic of local circuit neurons. This observation was confirmed in tissue immunolabeled for GABA, in which degenerating callosal terminals sometimes formed asymmetric synapses on GABA-labeled dendrites. In addition, GABA-labeled terminals and callosal afferents were sometimes observed to converge onto common postsynaptic dendritic shafts or spines within the PFC. These results indicate that callosal terminals in limbic association cortex, consistent with sensory and motor cortices, primarily target the spines of pyramidal neurons. In addition, the results suggest that callosal afferents to the PFC interact with GABA local circuit neurons at multiple levels. Specifically, a proportion of callosal terminals appear to provide excitatory drive to GABA cells, while GABA terminals may modulate the excitation from callosal inputs to the distal dendrites and spines of PFC pyramidal neurons. Synapse 29:193–205, 1998. © 1998 Wiley-Liss, Inc.     

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and γ-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.     

GABA-immunoreactive neurons in the nematode Ascaris

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.     

Direct demonstration of interactions between substance P immunoreactive terminals and tyrosine hydroxylase immunoreactive neurons in the substantia nigra of the rat: an ultrastructural study

The results of many anatomical, physiological, and pharmacological studies suggest that substance P-containing neurons of the striatum project to the substantia nigra, and that substance P influences the activity of dopaminergic nigrostriatal neurons. The purpose of the present ultrastructural study was to employ dual immunocytochemical labeling to determine the morphological basis for the observed actions of substance P on nigral dopaminergic neurons. Substance P-like and tyrosine hydroxylase-like immunoreactivities were localized simultaneously at the ultrastructural level in the substantia nigra of the rat. A double label method was utilized which relied on a combination of the peroxidase-antiperoxidase method (Sternberger, 1979) for substance P, and immunogold or silver enhanced immunogold labeling for tyrosine hydroxylase. The present results indicate that tyrosine hydroxylase immunoreactive (THLI) dendrites in the substantia nigra receive synaptic input from terminals exhibiting substance P-like immunoreactivity. These findings support the idea that substance P is a major neurotransmitter in the striatonigral loop, and suggest that striatal substance P neurons act directly upon nigral dopaminergic cells.     

Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals

The ultrastructure, afferent input, and sites of termination of neurons containing neuropeptide Y-like immunoreactivity (NPY-LI) were examined in the adult rat nucleus accumbens by using the peroxidase-antiperoxidase (PAP) method. The NPY-LI was seen in sparsely distributed, spindle-shaped perikarya having cross-sectional diameters of 15-20 microns. These perikarya exhibited highly invaginated nuclear membranes and thin rims of cytoplasm containing Golgi lamellae, dense-core vesicles, and other organelles. A few large, principally aspiny, dendrites also showed NPY-LI. The dendrites received synaptic input from unlabeled terminals forming both symmetric and asymmetric junctions. Immunolabeling for NPY was evident in other processes that were not clearly differentiated as dendrites or axons. These were seen primarily near glial processes and the basal laminae of blood vessels. A few myelinated and many unmyelinated axons and axon terminals also were labeled for NPY. These terminals contained numerous, small (40-60 nm), clear and one or more large (80-100 nm) dense core vesicles. Forty-seven percent (27 out of 57) of the terminals containing NPY-LI formed symmetric junctions with unlabeled dendrites or dendritic spines. The remainder lacked recognizable densities within single planes of section. The neurons exhibiting NPY-LI in the nucleus accumbens were characterized further with respect to their afferent input from terminals labeled for the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD). Immunogold labeling of a rabbit antiserum against NPY and PAP labeling for a sheep antiserum to GAD were sequentially applied to the same sections. The GAD-labeled terminals formed symmetric junctions primarily with the more numerous unlabeled dendrites. However, a few synaptic junctions also were detected between the GAD-labeled terminals and dendrites showing immunogold labeling for NPY. We conclude (1) that in the rat nucleus accumbens, NPY-LI is found principally in neurons of the aspiny type and (2) that the output from these presumably intrinsic neurons to other neighboring neurons or blood vessels is at least partially modulated by GABA.     

Serotonin-containing terminals synapse on septohippocampal neurons in the rat

Serotonin [5-hydroxytryptamine (5-HT)] is thought to be involved in mnemonic functions and dysfunctions possibly by directly contacting neurons in the medicl septal and diagonal band nuclei (i.e., the septal complex) that project to the hippocampal formation. However, there is no cellular substrate for this modulation. Thus, we examined the ultrastructure and synaptic association of 5-HT-containing terminals in relation to septohippocampal neurons in the septal complex of the rat brain. Projection neurons were identified by retrograde transport of wheat germ agglutinated apo-horseradish perodidase conjugated to colloidal gold particles (WANG) following an injection into the ventral hippocampal formation of uneashetized adult rats. After a 1 day survival, setions through the septal complex were labeled with antibodies to 5-HT immunoreactivity (5-HT-I) were observed in close proximity to neurons containing retogardely transported WAHG. By electron microscopy, 5-HT-I immunoreacitity (5-HT-I) were observed in close proximity to neurons containing retrogadely transported WAHG. By electron microscopy, 5-HT-I was found exclusively in axons and axon terminals. Axons were primarily unmyelinated. Terminals with 5-HT-I were 0.35–1.2 μm in diameter and contained numerous small, clear vesicles and 0–4 large, dense-core vesicles. The 5-HT-labeled terminals: (1) contacted perikarya and dendrites (220 of 349); (2) were closely apposed to other terminals (25 of 349); or (3) had no neuronal contacts in the plane of section analyzed (104 of 349). The 5-HT-labeled terminals formed exclusively symmetric synases on perikarya; some of these perikarya as well as some large dendrites similarly contacted by the 5-HT-labeled terminals also contained WAHG affiliated with lysosomes and multivesicular and “sequestration” bodies in the cytoplasm. However, the majority of terminals with 5-HT-I formed contacts on the shafts of small unlabeled dendrites (69% of 220); most of these were characterized as either asymmetric synapses or appositions not separated by astrocytes in the plane of section analyzed. We conclude that 5-HT-containing terminals in the rat septal complex: (1) directly modulate septohippocampal and other neurons through symmetric (potentially inhibitory) synapses on soma and proximal dendrites; and (2) form primarily asymmetric (potentially excitatory) synapses with distal (small) dendrites from neurons of unidentified origin. These findings suggest that serotonin may affect learning and memory through modulation of septal efferents to the hippocampal formation and may have direct relevance to the neuropathological basis for Alzheimer's disease. © 1993 Wiley-Liss, Inc.     

Influence of lateral septum and amygdala stimulation on the excitability of hypothalamic supraoptic neurons. An electrophysiological study in the rat

Extracellular recordings were obtained from 116 phasically-active (putative vasopressinergic) and 113 continuously-active (putative oxytocinergic) neurosecretory neurons in the hypothalamic supraoptic nucleus of urethane or pentobarbital anesthetized male Sprague-Dawley rats. Single 1 Hz pulse stimulation in most regions of the amygdala and the ipsilateral lateral septum was followed by a transient (20–140 ms) reduction in the excitability of more than 90% of responsive cells; one third displayed a reduction in excitability to both amygdala and lateral septum stimulation. Amygdala or lateral septum stimuli delivered in brief trains of 20–100 pulses at 5–20 Hz during ongoing phasic discharges could induce silent periods lasting to 30 or more seconds beyond the time of application. The same stimuli also reduced ongoing activity among continuously-firing SON cells but their response lasted only as long as the duration of the applied stimulus. These data suggest that neurons in both the ipsilateral lateral septum and the various amygdaloid nuclei exert a predominantly inhibitory influence on the excitability of both vasopressinergic and oxytocinergic SON neurons in the rat.     

Completely isolated molluscan neurons. An ultrastructural study.

The neurons of the molluscs Lymnaea and Helix isolated by fermentative digestion followed by mechanical treatment do not differ ultrastructurally from intact ones. These cells have sufficient metabolic reserves and incorporate into RNA 8% of the total radioactive pool, even more than neurons in ganglia under equal conditions. Neuronal damage can occur, mainly during the pipetting, and this is usually expressed in vacuolization of the cytoplasm. It is important to note that alterations in cell ultrastructure develop earlier than changes in the membrane electrical properties. The surface of the isolated neurons is enlarged two-fold due to the infoldings of the cell membrane. So, the specific resistance of soma membrane of these neurons was calculated as 78 +/- 13 komega-sq. cm. On the surface of isolated neurons scraps of glial and neuronal processes not connected with their own cell bodies, and as a consequence not powerful, are sometimes found. Some endings of the neuronal processes on the surface of isolated neurons are ultrastructurally similar to the axo-somatic synapses.     

GABA-immunoreactive neurons in the rat dorsal lateral geniculate nucleus: light microscopical observations

The dorsal lateral geniculate nucleus (dLGN) of the rat was investigated immunocytochemically using an antiserum against the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). The appearance of GABA-immunopositive dendrites, dendritic appendages, and the size and shape of neuronal somata closely resembled the putative intrinsic neurons described previously in Golgi-impregnation studies of the rat dLGN.     

An ultrastructural study of the red nucleus in the rat.

The red nuclei of 14 adult male rats of the Wistar strain were prepared for electron microscopic study following perfusion with a mixture of aldehydes, Neurons of four size categories were identified in 1 mu Epon sections and their ultrastructural characteristics were studied in adjacent thin sections. Giant (greater than 40 mu) and large (26-40 mu) neurons are distinguished primarily by size and possess similar ultrastructural features: extensive areas of rough endoplasmic reticulum (RER), a prominent perinuclear Golgi complex, numerous mitochondria and pigment granules and a large, ovoid nucleus which occasionally contains intranuclear rodlets. Medium size neurons (20-25 mu) have less extensive, poorly organized RER and randomly distributed Golgi complexes. The nuclear envelopes of these cells frequently show multiple invaginations and continuity with the RER cisternae. In small neurons (less than 20 mu) the RER occurs as single or anastomosing strands whi le golgi complexes and pigment granules are few. In both medium size and small neurons, aggregates of condensed chromatin are adherent to the inner nuclear membrane. Three main types of synaptic terminals may be distinguished in the red nucleus: (1) small terminals with flattened vesicles and symmetrical densities (F terminals), (2) small terminals with rounded vesicles and asymmetrical densities (RS terminals), and (3) large (10-15 mu) asymmetrical, rounded vesicle terminals which form multiple contacts along their length (RL terminals). The small neurons receive both F and RS terminals on their dendrites and infrequently on their cell somas. The large and giant neurons receive F, RS and RL terminals on their somas and proximal dendrites and F and RS terminals on their distal dendrites. The somas and dendrites of medium size neurons receive both F and RS terminals but RL terminals do not lie in relation to them. Spine contacts are common throughout the nucleus and occur on both somas and dendrites.     

An ultrastructural study of dynorphin-immunoreactive nerve fibers and terminals in the celiac-superior mesenteric ganglion of the guinea pig

Dynorphin-immunoreactive nerve fibers and terminals were identified in the celiac-superior mesenteric ganglion of the guinea pig at the ultrastructural level with the peroxidase-antiperoxidase technique. The immunostained material was localized in the large dense core vesicles of the terminals but was also present diffusely in the axoplasm. The terminals formed numerous axodendritic and a few axosomatic contacts, interpreted as synapses, with the principal ganglion cells. These findings suggest that dynorphin plays a role as a neurotransmitter or neuromodulator in the ganglion and, taken together with earlier findings, indicate an involvement of dynorphin neurons in the intestino-intestinal inhibitory reflex.     

GABA-immunoreactive neurons in the mushroom bodies of the honeybee: an electron microscopic study

Synaptic contacts of gamma-aminobutyric acid (GABA) -immunoreactive neurons in honeybee mushroom bodies were studied by using electron microscopic immunocytochemistry. In the lip region of the calyx neuropil, GABA-immunoreactive profiles formed synapses onto both small postsynaptic profiles (76%) and large immunonegative boutons (4%), which were likely to belong to the intrinsic and extrinsic mushroom body neurons, respectively. Three morphologic types of the large immunonegative boutons were distinguished: "light," "dark," and "dense core"; all of them received synaptic inputs from the GABA-immunoreactive profiles. A significant proportion of the synapses formed by the GABA-immunoreactive neurons in the lip region (20%) were input synapses from immunonegative neurons. Analysis of thin serial sections showed that the output and input synapses formed microcircuits in which both large immunonegative boutons and small postsynaptic profiles were involved. We interpret these findings to show that negative feedforward and feedback loops exist within the microcircuits of the lip region.     

Axon terminals immunolabeled for dopamine or tyrosine hydroxylase synapse on GABA-immunoreactive dendrites in rat and monkey cortex

Dopamine afferents to the cortex regulate the excitability of pyramidal neurons via a direct synaptic input. However, it has not been established whether dopamine also modulates pyramidal cell activity indirectly through synapses on γ-aminobutyric acid (GABA) interneurons, and whether such inputs differ across cortical regions and species. We sought to address these issues by an immunocytochemical electron microscopic approach that combined peroxidase staining for dopamine or tyrosine hydroxylase (TH) with a pre-embedding gold-silver marker for GABA. In the deep layers of the rat prefrontal cortex and in the superficial layers of the monkey prefrontal and primary motor cortices, terminal varicosities immunoreactive for dopamine or TH formed primarily thin, symmetric synapses on distal dendrites. Both GABA-immunoreactive dendrites as well as unlabeled spines and dendrites were contacted by dopamine- or TH-immunoteactive terminals. Synaptic specializations were detected at some, but not all of these contacts. The relative frequency of these appositional and synaptic contacts did not appear to differ between the rat and monkey prefrontal cortex, or between the monkey prefrontal and motor cortices. Across regions and species, labeled and unlabeled targets of dopamine- or TH-positive terminals received additional synaptic input from unlabeled, and occasionally GABA-immunoreactive terminals. Close appositions between dopamine- or TH immunoreactive and GABA-positive terminals were observed only rarely. These findings indicate that dopamine afferents provide direct synaptic inputs to GABA local circuit neurons in a consistent fashion across cortical regions and species. Thus, dopamine's cellular actions involve direct as well as modulatory effects on both GABA interneurons and pyramidal projection neurons. © 1995 Wiley-Liss, Inc.     

Glycine-immunoreactive synaptic terminals in the nucleus tractus solitarii of the cat: ultrastructure and relationship to GABA-immunoreactive terminals.

Postembedding immunogold labeling methods applied to ultrathin and semithin sections of cat dorsomedial medulla showed that neuronal perikarya, dendrites, myelinated and nonmyelinated axons, and axon terminals in the nucleus tractus solitarii contain glycine immunoreactivity. Light microscopic observations on semithin sections revealed that these immunoreactive structures were unevenly distributed throughout the entire nucleus. At the electron microscopic level, synaptic terminals with high levels of glycine-immunoreactivity, assumed to represent those releasing glycine as a neurotransmitter, were discriminated from terminals containing low, probably metabolic levels of glycine-immunoreactivity, by a quantitative analysis method. This compared the immunolabeling of randomly sampled terminals with a reference level of labeling derived from sampling the perikarya of dorsal vagal neurones. The vast majority of these "glycinergic" terminals contained pleomorphic vesicles, formed symmetrical synaptic active zones, and targeted dendrites. They appeared to be more numerous in areas of the nucleus tractus solitarii adjoining the tractus solitarius, but rather scarce caudally, medially, ventrally, and in the dorsal motor vagal nucleus. In a random analysis of the entire nucleus tractus solitarii, 26.2% of sampled terminals were found to qualify as glycine-immunoreactive. In contrast, boutons immunoreactive for gamma-aminobutyric acid (GABA) were more evenly distributed throughout the dorsal vagal complex and accounted for 33.7% of the synaptic terminals sampled. A comparison of serial ultrathin sections suggested three subpopulations of synaptic terminals: one containing high levels of both GABA- and glycine-immunoreactivities (21% of all terminals sampled), one containing only GABA-immunoreactivity (12.7%), and relatively few terminals (5.2%) that were immunoreactive for glycine alone. These results were confirmed by dual labeling of sections using gold particles of different sizes. This study reports the first analysis of the ultrastructure of glycinergic nerve terminals in the cat dorsal vagal complex, and the pattern of coexistence of glycine and GABA observed provides an anatomical explanation for our previously reported inhibitory effects of glycine and GABA on neurones with cardiovascular and respiratory functions in the nucleus tractus solitarii.