NADPH-diaphorase-positive neurons in the human inferior colliculus: morphology, distribution and clinical implications.

Using the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction with nitroblue tetrazolium, we provided a detailed investigation of the distribution, dimensional characteristics and morphology of NADPH-d-positive neurons in the three main subdivisions of the human inferior colliculus (IC): central nucleus, pericentral nucleus, and external nucleus. In accordance with their perikaryal diameter, dendritic and axonal morphology, these neurons were categorized as large (averaging up to 45 µm in diameter), medium (20-30 µm), small (13-16 µm) and very small (7-10 µm). Their morphological differences could contribute to varying functionality and processing capacity. Our results support the hypothesis that large and medium NADPH-d-positive cells represent projection neurons, while the small cells correspond to interneurons. Heretofore, the very small NADPH-d-positive neurons have not been described in any species. Their functions-and if they are, indeed, the smallest neurons in the IC of humans-remain to be clarified. Owing to their location, we posit that they are interneurons that connect the large NADPH-d-positive neurons and thereby serve as an anatomical substrate for information exchange and processing before feeding forward to higher brain centers. Our results also suggest that the broad distribution of nitric oxide (NO) synthesis in the human IC is closely tied to the neuromodulatory action of NO on collicular neurotransmitters such as GABA and glutamate, and to calcium-binding proteins such as parvalbumin. A deeper understanding of the relationship between NADPH-d-positive fibers in all IC connections and their co-localization with other neurotransmitters and calcium-binding proteins will assist in better defining the function of NO in the context of its interplay with the cerebral cortex, the sequelae of the aging process and neurodegenerative disorders.

Neuropeptide Y immunoreactivity in the cat claustrum: A light- and electron-microscopic investigation.

The claustrum is a telencephalic nucleus located ventrolateral to the basal ganglia in the mammalian brain. It has an extensive reciprocal connectivity with most if not all of the cerebral cortex, in particular, primary sensory areas. However, despite renewed and growing interest amongst investigators, there remains a paucity of data concerning its peptidergic profile. The aim of the present study was to examine the presence, morphology, distribution and ultrastructure of neuropeptide Y-immunoreactive (NPY-ir) neurons and fibers in the claustrum of the cat. Ten adult healthy cats from both sexes were used. All animals received human and ethical treatment in accordance with the Principles of Laboratory Animal Care. Subjects were irreversibly anesthetized and transcardially perfused with fixative solution containing glutaraldehyde and paraformaldehyde. Brains were promptly removed, postfixed and sectioned. Slices were incubated with polyclonal anti-NPY antibodies according to the standard avidin-biotin-peroxidase complex method adopted by our Department of Anatomy, Histology and Embryology. NPY-ir neurons and fibers were found to be diffusely distributed throughout the claustrum, with no obvious topographic or functional patterning other than larger numbers in its central/broadest part (stereotaxic planes A12-A16). Neurons were generally classified by diameter into three sizes: small (under 17 µm), medium (17-25 µm) and large (over 25 µm). Staining density is varied with some neurons appearing darker than others. At the electron-microscopic level NPY immunoproduct was observed within neurons, dendrites and terminal boutons, each differing relative to their ultrastructural attributes. Two types of NPY-ir synaptic boutons were found. Lastly, it is of interest to note that gender-specific differences were not observed.

Parvalbumin-immunoreactive neurons in the human claustrum.

The morphology and distribution of parvalbumin-immunoreactive neurons (PV-ir) were studied in the human claustrum. PV-ir neurons were observed throughout the claustrum, with the highest numbers noted in the central (broadest) portion as compared with the dorsal and ventral aspects. Reaction product was evident in the neuronal perikarya, dendritic processes, and spines. In the majority of these labeled neurons, the cytoplasm was devoid of lipofuscin pigment. Cell bodies varied widely in both shape and size, ranging from oval and small, to multipolar and large. PV-ir neurons were classified into two groups, primarily based on dendritic morphology: spiny neurons with long and straight dendrites, and aspiny neurons with thin and curving dendritic processes. PV-ir fibers were seen throughout the neuropil, with many immuno-positive puncta noted.

Imbalance of immunohistochemically characterized interneuron populations in the adolescent and adult rodent medial prefrontal cortex after repeated exposure to neonatal separation stress.

Experimental studies in various animal models have revealed convincing evidence that stressful experience during early developmental periods produces a variety of behavioral, neuroanatomical and endocrine alterations, which are reminiscent of human mental disorders such as depression and various types of anxiety disorders. Since these mental disorders are assumed to be associated with altered GABAergic inhibition in cortical and subcortical brain regions, the current study tested the hypothesis that early postnatal adverse emotional experience (separation stress) interferes with the establishment and functional maturation of distinct inhibitory interneuron populations in different subregions of the medial prefrontal cortex (mPFC) of the precocious rodent degu (Octodon degus). At the age around puberty early stressed animals displayed significantly lower densities of calbindin-D28k-immunoreactive interneurons in the anterior cingulate (down to 79%) and in the precentral medial (down to 64%) subregions of the mPFC compared with age-matched unstressed controls. At this age the densities of two other interneuron types characterized by their expression of the calcium-binding proteins parvalbumin or calretinin remained at control levels. In adulthood, i.e. after an extended period without stress exposure, the density of calbindin-D28k-immunoreactive interneurons in the stressed animals was back to control numbers, whereas parvalbumin-immunoreactive interneurons displayed significantly elevated density in the anterior cingulate (up to 138%) and in the precentral medial cortex (up to 137%) of the stressed animals. In both age groups the density of calretinin- and corticotropin releasing hormone-immunoreactive interneurons did not differ between stressed and control animals, and the prelimbic and infralimbic subregions of the medial prefrontal cortex remained unaffected by stress experience. These results confirm that early adverse emotional experience induces long lasting age-, region- and neuron-specific imbalance of inhibitory systems in some, but not all subregions of the medial prefrontal cortex of the degu.

Neuronal nitric oxide synthase immunopositive neurons in cat vestibular complex: a light and electron microscopic study.

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless, there are little data about the neuronal nitric oxide synthase immunoreactivity (nNOS-ir) in the vestibular complex of a cat. In this respect, the aims of this study were to: (1) demonstrate nNOS-ir in the neurons and fibers, from all major and accessory vestibular nuclei; (2) describe their light microscopic morphology and distribution; (3) investigate and analyze the ultrastructure of the NOS I-immunopositive neurons, fibers, and synaptic boutons. For demonstration of the nNOS-ir, the peroxidase-antiperoxidase-diaminobenzidin method was applied. Immunopositive for nNOS neurons and fibers were present in all major and accessory vestibular nuclei. On the light microscope level, the immunopositive neurons were different in shape and size. According to the latter, they were divided into four groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), large type I (with diameter from 30 to 40 microm), and large type II (with diameter greater than 40 microm). On the electron microscope level, the immunoproduct was observed in neurons, dendrites, and terminal boutons. According to the ultrastructural features, the neurons were divided into three groups--small (with diameter less than 15 microm), medium-sized (with diameter from 15 to 30 microm), and large (with diameter greater than 30 microm). At least two types of nNOS-ir synaptic boutons were easily distinguished. As a conclusion, we hope that this study will contribute to a better understanding of the functioning of the vestibular complex in cat and that some of the data presented could be extrapolated to other mammals, including human.

Juvenile emotional experience alters synaptic inputs on pyramidal neurons in the anterior cingulate cortex.

Analogous to the experience-driven development of sensory systems, the functional maturation of limbic circuits is significantly influenced by early socio-emotional experience. In a combined light and electron microscopic study in the anterior cingulate cortex of Octodon degus, the densities of spine and shaft synapses on apical dendrites of layer III pyramidal neurons were compared in 45 day old (1) undisturbed control animals; (2) handled animals; (3) animals which were repeatedly maternally deprived during the first three postnatal weeks; (4) animals which were treated similarly to group 3 and thereafter kept in chronic social isolation. Animals in groups 2-4 showed significantly higher spine densities (up to 121%, 142% and 151% respectively) compared to control group 1. Group 3 displayed significantly longer apical dendrites compared to control group 1. The electron microscopic analysis in cortical layer II revealed significantly higher spine synapses in group 4 (up to 166%) and fewer shaft synapses in groups 3 and 4 (down to 53% and 65% respectively) compared to group 1. These results demonstrate that early traumatic emotional experience alters synaptic input of pyramidal neurons. Such experience-induced modulation of limbic cortex development may determine psychosocial and cognitive capacities during later life.

Maternal separation and social isolation modulate the postnatal development of synaptic composition in the infralimbic cortex of Octodon degus.

We analysed the influence of preweaning periodic maternal separation followed by postweaning chronic social isolation on the development of synaptic composition in the infralimbic cortex of Octodon degus, a South American species formerly classified as a caviomorph rodent but now considered to belong to Lagomorpha (rabbits). Three groups of animals were analysed: (1) control pups which remained undisturbed with their families; (2) pups which were exposed to individual periodic maternal deprivation [postnatal day 1 (P1) until P21], followed by social isolation (P22 until P45); and (3) pups which were handled daily without being removed from the families (P1 until P21) and thereafter remained undisturbed with the family (P22 until P45). The mean synaptic density and mean projected height of synapses were quantified using the "dissector" method. In the deprived group, significantly higher (up to 137.8%) mean synaptic densities were found in layer II of the infralimbic cortex compared to normal control animals. In handled pups, asymmetric shaft synapses were significantly decreased (down to 54%) compared to the control group.These results indicate that early postnatal changes in the socio-emotional environment change the number of synaptic connections in the infralimbic cortex. Since this subregion of the medial prefrontal cortex is involved in a variety of emotional behaviors and plays a role in associative learning tasks, these environmentally induced synaptic changes may be indicative, and perhaps the cause, of alterations of behavioral and cognitive capacities.

Sexual dimorphism of the bed nucleus of the stria terminalis and the amygdala.

The present review summarizes the up-to-date knowledge on the sexual dimorphism of the CNS with special regard to the sexual differentiation of the bed nucleus of the stria terminalis (BST) and the amygdala in rat. The authors provide new evidence for the sexual dimorphism and differentiation of GABAergic, leucine-enkephalin-containing and parvalbumin-immunoreactive neurons in the BST and the amygdala of the rat. Together with testing the gender differences, age-related changes in numbers of the neuronal subpopulations, mentioned above are followed. The authors' results provide morphological and immunocytochemical data that may be used for further studies on sexually dimorphic circuitry and its functional significance.

The inferior oilvary complex.

Although many studies have established the connections of the inferior olivary complex, there have been relatively few studies on the morphology of this nuclear complex. On the base of the similar topographic relations and connections with the cerebellum, the inferior olivary complex is regarded as homologous in all vertebrates. We present comprehensive light microscopical qualitative and quantitative analysis of the inferior olivary complex in different representatives of submammilian and mammilian vertebrates, including human. A detailed comparison was made at the structural level. Cytoarchitecture and cellular morphology of the inferior olivary complex have been studied in several submammilian (carp, frog, lizard, tortoise, pigeon) and mammilian species (rat, cat, and for the first time ground squirrel Citellus citellus L), including human.

Intramembranous structure of synaptic membranes with special reference to spinules in the rat sensorimotor cortex.

The intramembranous structure of the synaptic contact zone at presynaptic and postsynaptic membranes in the rat sensorimotor cortex was examined by means of the freeze-etching technique. In axospinous synapses, the synaptic contact zone is characterized by perforated and nonperforated aggregates of intramembranous particles at the extracellular half or E-face of the postsynaptic membrane. On some perforated synaptic contact zones, both synaptic membranes are marked by so called spinules. These invaginations of the postsynaptic membrane and the parallel presynaptic membrane into the axon terminal are situated at the particle free zones among the postsynaptic E-face intramembranous particle aggregates or in close proximity to it. Intramembranous characteristics of the spinules at both freeze-etched faces of presynaptic and postsynaptic membranes and their density of perforated axospinous synapses were analysed. The results are discussed in terms of plasticity at the synaptic contact zone of the axospinous synapses of the sensorimotor cortex in the rat.

Characterization of vesicular membrane-bound alpha-SNAP and NSF in adrenal chromaffin cells.

alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg2+ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant his6-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Our data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.

Sex and age differences of neurons expressing GABA-immunoreactivity in the rat bed nucleus of the stria terminalis.

Neurons, containing GABA were visualised immunohistochemically in the bed nucleus of the stria terminalis. Young prepubertal (20 days of age) and postpubertal (3 months and 1 year of age) Sprague-Dawley rats were used. Quantitative studies revealed greater density of GABA-immunoreactive perikarya in female than in male bed nucleus of the stria terminalis. This difference was not due to distribution in different volumes, since the volumes of the bed nucleus of the stria terminalis in the three ages studied did not differ by gender. Castration of new-born male rats caused elevation of the density of GABA-immunoreactive neurons in the bed nucleus of the stria terminalis to female levels on the third month of life. The percentage of nerve cells, expressing detectable amounts of GABA increased with age in the rat bed nucleus of the stria terminalis. The sexual dimorphism of GABA-immunoreactive neurons in the bed nucleus of the stria terminalis may contribute to the formation of reproductive behavior. The elevation of GABA expression with age might reflect change of the cellular activity in this part of the limbic circuitry.

Evidence for a possible glycinergic inhibitory neurotransmission in the midbrain and rostral pons of the rat studied by gephyrin.

The data on the glycinergic transmission in the rostral brainstem are both few and controversial. The present report provides evidence for a possible glycinergic transmission in Sprague-Dawley rats, based on observations of immunocytochemical labeling for gephyrin, a 93 kDa protein and a component of the functional glycine receptor. A monoclonal antibody against gephyrin was used, and the reaction product was visualized by means of avidin-biotin-peroxidase procedure. The reaction product in midbrain and rostral pons was found in neuronal perikarya and in proximal dendrites but in some cases the most distal dendritic branches were also labeled. The neuropil usually displayed a moderate staining with finely granulated reaction product. The most significant immunocytochemical signal was mainly encountered in large and medium-sized neuronal populations of the motor cranial nerve nuclei (III, IV, V), in the reticular formation (laterodorsal tegmental nucleus, pedunculopontine tegmental nucleus, deep mesencephalic nucleus), in the red nucleus, in the intermediate and deep gray strata of the superior colliculus. Only in the substantia nigra and the inferior colliculus the parvocellular cell populations were mainly labeled. The present data suggest a significant inhibitory glycinergic neurotransmission in the rostral brainstem, probably mediated by interneurons.

Topographical distribution of NADPH-diaphorase-positive neurons in the cat's claustrum.

The NADPH-diaphorase histochemical technique was used to visualize the morphological features of the NADPH-diaphorase positive cells and fibres, and their distribution in both parts of the cat's claustrum. Taking into account the size and form of the perikaryon and the dendritic and axonal characteristics, the neurons are grouped in different subclasses: large, medium-sized and small. The present data suggest the occurrence of two populations of NADPH-diaphorase neurons in the claustrum. One population consisting of large and medium-sized positive neurons represents the projection neurons while the other population of small positive neurons corresponds to the local circuit neurons.

Distribution of GABA-immunoreactive nerve cells in the bed nucleus of the stria terminalis in male and female rats.

The distribution of GABA-immunoreactive neurons in the subnuclei of the rat bed nucleus of the stria terminalis (BST) was studied by means of GABA immunohistochemistry. For detection of GABA immunoreactivity we used polyclonal antibodies and silver intensification. We have compared the pattern of distribution of immunoreactive cells in male and female rats and found some sexual difference, that may underlie functional variety. Computer assisted quantitative analysis of GABA-immunoreactive neurons per mm2 showed statistically significant sex differences in the medial part of the BST (0.001 < P < 0.01). The difference in the BST as a whole was set at 0.05 < P < 0.1. Females had more numerous GABA-immunoreactive cells than males. The measuring of sex differences was done using double-tailed Student's t-test after submitting the data to ANOVA.

Structure of the synaptic junctions in the rat sensorimotor cortex. Freeze-etching study of axodendritic synapses.

The intramembranous structure of axodendritic synapses in the rat sensorimotor cortex was studied by means of freeze-etched replicas and thin sections. In thin sections, symmetric synaptic junctions were located on dendritic shafts. Examination of freeze-etched preparations supported the notion that the postsynaptic dendritic shaft membrane exhibited the same structure as the surrounding non-junctional membrane on both fracture faces. In thin sections, asymmetric perforated and non-perforated synapses were found on dendritic spines and dendritic shafts. In freeze-etched replicas, the postsynaptic membrane of the dendritic spine and some of the dendritic shaft synapses were characterized by perforated and non-perforated aggregates of intramembranous particles at the extracellular half (E-face). These aggregates are accepted as a freeze-etch equivalent of perforated and non-perforated asymmetric synapses seen in thin sections. The intramembranous characteristics of axodendritic synapses in the rat sensorimotor cortex are discussed in terms of synaptic contact zone plasticity.

Intramembranous structure of the postsynaptic membrane in the rat sensorimotor cortex.

Intramembranous structure of the postsynaptic membrane of the axodendritic synapses in the rat sensorimotor cortex was examined by means of freeze-etching technique. Perforated and non-perforated aggregates of particles were found at the extracellular half (E-face) of the postsynaptic membrane. To study correlation between membrane structure and synaptic plasticity we compared size and particle packing density in both type aggregates of particles at the E-face of the postsynaptic dendritic spine membrane. The results are discussed in terms of plasticity on the synaptic contact zone (SCZ) at the postsynaptic membrane of the excitatory axospinous synapses.

Structure of the synaptic junctions in the rat sensorimotor cortex: freeze-etching study of neuronal gap junctions.

The membrane structure of neuronal gap junctions in the rat sensorimotor cortex was examined using freeze-etched replicas. The gap junction coupling was found between large, probably non-pyramidal neurons and dendrites, and was mostly associated with membrane specializations indicative of the contact zone of an asymmetric synapse or a puncta adhaerentia. The validity of freeze-etching method of identification and analysis of neuronal gap junctions as a feature of the synaptic organization in the rat sensorimotor cortex is discussed.

Light and electron microscopic demonstration of cholesterol distribution in membrane structures of the rat auditory cortex.

As part of our investigations on the changes in the cortex of different stages of ontogeny, the aim of this study was to analyse the cholesterol distribution in the auditory cortex of adult rats. The light microscopic Schultz reaction as well as electron microscopic thin sections and freeze-etching combined with a cholesterol specific marker were applied to cholesterol demonstration. High and low cholesterol areas were found in the plasma membrane and membranes of some organelles. A low cholesterol content was observed in the membranes of the Golgi apparatus, mitochondria and junctional contacts. A very low cholesterol content was found in the pre- and postsynaptic membranes. High cholesterol contents were present in the neuronal and glial non-junctional plasma membranes. The cholesterol distribution in the membranes of the endoplasmic reticulum and nuclear envelope appeared to be different.