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A laminar organization composed of the dendritic fields of principal neurons and afferent axonal arbors has been proposed as the anatomical substrate for the frequency map at several levels of the mammalian central auditory system, including the inferior colliculus and medial geniculate body (MGB). In contrast to the auditory thalamus in most mammals, the ventral division of the rabbit medial geniculate body (MGV) has cellular laminae visible in routine Nissl stains, allowing a direct comparison of the laminar organization with the dendritic architecture and frequency organization. In total 30 presumptive relay neurons in the MGV were labeled with the juxtacellular recording method, and their dendritic arbors were fully reconstructed from serial sections with the aid of a computer microscope. The spatial organization of MGV dendritic fields was analyzed using the dendritic prism, dendritic stick, and fan-in projection methods. Quantitative spatial analyses revealed that, for MGV neurons in the central pars lateralis subdivision, the major axis of the dendritic fields (approximately 29 degrees relative to the horizontal plane) was closely aligned with that of the Nissl laminae (approximately 25 degrees). Both were oriented orthogonally to the tonotopic axis. In contrast, cells in the pars ovoidea had their major axis of orientation parallel to the anteroposterior axis of the brain. Although a bitufted dendritic field was the norm, it was not uncommon for MGV neurons to have pronounced spatial asymmetries in their dendritic fields. A model is presented that incorporates cellular laminae and oriented dendritic growth to form frequency-related slabs within the MGV., (Copyright 2003 Wiley-Liss, Inc.)

Although there is evidence for multiple classes of thalamic relay neurons in the auditory thalamus, correlative anatomical and physiological studies are lacking. We have used the juxtacellular labeling technique, in conjunction with Nissl, Golgi, and immunocytochemical methods, to study the morphology and response properties of cells in the ventral division of the medial geniculate body of the rabbit. Single units in the ventral division of the medial geniculate body (MGV) were characterized extracellularly with monaural and binaural tone and noise bursts (100- to 250-msec duration). Characterized units were filled with biocytin and visualized with an antibody enhanced diaminobenzidine reaction. A total of 31 neurons were physiologically characterized and labeled with the juxtacellular technique. Labeled neurons were fully reconstructed from serial sections by using a computer microscope system. Three subregions of the rabbit MGV were identified, each characterized by differences in Nissl architecture, calcium-binding protein expression, and by the dendritic orientation of tufted relay neurons. In general, the dendritic fields of relay neurons were closely aligned with the cellular laminae. Qualitative and quantitative analyses revealed two types of presumptive relay neurons within the MGV. Type I cells had thick dendrites with a greater total volume and morphologically diverse appendages compared with the Type II cells whose dendrites were thin with a moderate number of small spines. Both classes were acoustically responsive and exhibited a variety of response patterns, including onset, offset, and sustained responses. In terms of binaural characteristics, most (ca. 53%) labeled neurons were of the EE type, with the remaining cells classified as EO (27%) or EI (20%) response types. Two types of presumptive interneurons were also seen: bipolar neurons with large dendritic fields and a small neurogliaform variety. Cell types and dendritic orientation within the MGV are discussed in terms of the physiological organization of the rabbit auditory thalamus., (Copyright 2002 Wiley-Liss, Inc.)

Recent studies of the rabbit auditory forebrain have shown that antibodies directed against the calcium-binding protein parvalbumin (PV) specifically demarcate auditory neocortex and the ventral division of the medial geniculate body (MGV). The auditory cortex is characterized by two PV- immunoreactive bands: dense terminal-like labeling within layer III/IV and a prominent band of PV+ somata in the upper half of layer VI. In some cases, there are distinct patches of PV immunoreactivity within layers III/IV of auditory cortex that appear similar to the patchy termination of thalamocortical axons labeled by the injection of anterograde tracers into MGV. The presence of PV+ patches in III/IV, PV+ somata in layer VI, and the high density of PV+ neurons and terminals in the MGV suggest the existence of a reciprocal PV+ circuit linking primary auditory cortex (AI) and the MGV. In the present study, double-labeling experiments in adult rabbits were carried out to provide evidence for this circuit. Focal injections of the tracers biocytin or biotinylated dextran amine (BDA) into the MGV labeled thalamocortical afferent patches within layer III/IV and retrogradely labeled corticothalamic neurons in layer VIa of the ipsilateral auditory cortex. Adjacent sections stained with antibodies against PV revealed terminal-like PV-immunoreactive patches in III/IV and PV+ somata in VIa that were in register with those labeled by BDA injections into the MGV. Serial section reconstruction of BDA-labeled corticothalamic neurons in VIa revealed pyramidal cells with tangentially oriented basal dendrites and sparsely branched apical dendrites that ascended to layer I. Fluorescent double-labeling studies demonstrated that a subpopulation of corticothalamic neurons also express PV. PV-negative corticothalamic neurons were also found. Discrete injections of BDA into auditory cortex labeled bands of neurons in the ipsilateral MGV, whose orientation paralleled the fibrodendritic laminae characteristic of this subdivision. Retrograde double-labeling experiments showed that most MGV relay neurons also express PV. Small numbers of PV-negative relay neurons were also found. These studies provide evidence for the existence of multiple, chemically coded pathways linking primary auditory cortex and the MGV.

Recent studies have shown that changes in dendritic architecture are an important component of functional plasticity in the adult central nervous system. In the present study, we determined whether gonadectomy induces changes in dendritic architecture in the arcuate nucleus, a target tissue for gonadal hormones. A combination of retrograde labeling with systemically injected Fluoro-Gold and intracellular injection of neurons in a fixed-slice preparation was used to examine the morphology of neuroendocrine neurons in the rat arcuate nucleus. Intracellullary filled arcuate neuroendocrine neurons (8-21 neurons per brain) from intact (n = 5) and orchidectomized (n = 5) animals were reconstructed with the aid of a computer microscope. A quantitative analysis revealed that orchidectomy had no effect on the number and distribution of Fluoro-Gold-labeled neuroendocrine neurons in the rat arcuate nucleus. The morphology of arcuate neuroendocrine neurons in intact animals was relatively simple, with the majority of neurons (79%) having only two primary dendrites and few dendritic spines. Compared with intact controls, arcuate neuroendocrine neurons in the orchidectomized group had significantly larger somatic profile areas and exhibited significant increases in dendrite length, dendrite volume, terminal branch number, and spines per unit length of dendrite. The increase in terminal branch number in orchidectomized animals was due primarily to the appearance of short branches that gave a striking, claw-like appearance to many of the distal dendrites. These results provide evidence for hormonal regulation of dendritic morphology of arcuate neuroendocrine neurons in adult mammals.

In situ hybridization histochemistry was used to map the distribution of neurons expressing the substance P (SP) or neurokinin B (NKB) genes in the human hypothalamus and basal forebrain. Hypothalami from five adult males were frozen in isopentane at -30 degrees C and serially sectioned at 20 jm thickness. Every 20th section was hybridized with [35S]-labeled, 48-base synthetic cDNA probes that were complementary to either SP or NKB mRNAs. Slides were dipped into nuclear emulsion for visualization of mRNAs at the single-cell level. The location of labeled neurons (greater than x 5 background) was mapped by using an image-combining computer microscope system. A distinct and complementary distribution pattern of SP and NKB neurons was observed in the human hypothalamus and basal forebrain. NKB was the predominant tachykinin in the rostral hypothalamus, whereas SP mRNA predominated in the posterior hypothalamus. Numerous NKB neurons were identified in the magnocellular basal forebrain, the bed nucleus of stria terminalis, and the anterior hypothalamic area. Scattered NKB neurons were present in the infundibular and paraventricular nuclei, paraolfactory gyrus, posterior hypothalamic area, lateral division of the medial mammillary nucleus, and amygdala. Numerous neurons expressing SP mRNAs were identified in the premammillary, supramammillary, and medial mammillary nuclei; the posterior hypothalamic area; and the corpus striatum. Scattered SP neurons were also observed in the preoptic area; the infundibular, intermediate, dorsomedial, and ventromedial nuclei; the infundibular stalk; the amygdala; the bed nucleus of stria terminalis; and the paraolfactory gyrus. These studies provide the first description of the location of neurons that express tachykinin gene transcripts in the human hypothalamus.

The complementary pattern of immunohistochemical staining for the calcium-binding proteins parvalbumin (PV) and calbindin D-28k (CB) was used to delineate four major subdivisions of the rabbit medial geniculate body (MGB). PV immunoreactivity predominates in the ventral and medial divisions, whereas CB-immunoreactive cells characterize the dorsal and internal divisions. The ventral nucleus is strongly PV+ due to dense neuropil labeling and moderately labeled somata. The medial nucleus contains both medium-sized and large PV+ somata, as well as thick PV+ axons and terminals. The wedge-shaped internal nucleus composed of densely labeled CB+ cells, separates the dorsal and ventral nuclei rostrally, and expands caudally to encapsulate the posterior MGV. Large multipolar CB+ neurons with radiate dendrites characterize the dorsal nucleus. The differential expression of PV and CB also distinguishes the deep dorsal and superficial dorsal subnuclei in the dorsal division and a ventrolateral component in the ventral division. A comparison with studies of MGB connectivity in a variety of species suggests that PV immunoreactivity is highest in subdivisions that receive a substantial input from the central nucleus of the inferior colliculus and that project to primary auditory cortex. In contrast, CB immunoreactivity characterizes nuclei that receive input primarily from other sources, such as the paracentral nuclei of the inferior colliculus, the lateral tegmentum, and the spinal cord, and that project to secondary auditory areas. The ability of calcium-binding protein immunohistochemistry to delineate neuronal compartments across indistinct cytoarchitectonic borders makes it a powerful tool for guiding future connectional and physiological studies of the MGB.

Parvalbumin (PV) is a calcium-binding protein present in GABAergic cells in the cerebral cortex and in thalamic relay neurons. In the present study, parvalbumin immunocytochemistry (PVi) and stereological methods were used to obtain estimates of cortical volume, total neuron number, laminar density, and the percentage of PV-immunoreactive neurons in auditory neocortex. PVi clearly delineated the primary auditory cortex (AI), which was characterized by two PV+ bands: dense terminal-like labeling within lamina III/IV and PV+ somata in lamina VIa. Stereological analysis of Nissl-stained sections revealed that the total number of neurons in rabbit AI was 1.48 x 10(6) with a mean neuronal density of 55 x 10(3)/mm3. Based on a mean cortical thickness of 1.92 mm, there are approximately 106,000 neurons in a 1 mm2 column of auditory cortex. PVi yields an extraordinary Golgi-like staining of nonpyramidal cells in all cortical layers. PV+ nonpyramidal cells constitute approximately 7.0% of the neurons in AI. There were significant differences in the morphology and density of PV+ neurons across layers. Although only 5% of cells in lamina I were PV+, three nonpyramidal cell types were present. Lamina II had the highest numerical density within AI but the lowest percentage of PV+ neurons (3.3%). Lamina II, however, contained the greatest diversity of PV+ nonpyramidal cell types, which included small multipolar cells, bipolar cells, and, less frequently, large cells of the bitufted, bipolar, and stellate varieties. Lamina IV had one of the highest numerical densities (67.6 x 10(3) neurons/mm3) and contributed nearly 27% of the total neuron number in AI. The numerical density of PV+ nonpyramidal cells was also greatest within lamina IV (7.1 x 10(3)/mm3) where they formed 10.4% of the neuronal population. PV+ nonpyramidal cells in lamina IV and lamina III were predominantly large basket-type cells with bitufted dendritic domains and tangentially oriented local axonal plexuses. The terminal-like label within lamina III/IV derived in part from the basket-cell axons, which formed pericellular arrays around unstained somata. Cell-sparse lamina V contained the largest PV+ nonpyramidal cells in AI. These cells, which formed 11% of the neuron population in lamina V, were notable for their tangentially oriented dendritic fields and local axonal arbors. PVi partitioned lamina VI into VIa and VIb. Large multipolar nonpyramidal cells were distributed throughout lamina VI and made up approximately 6% of the total population. Lamina VIa contained a band of lightly labeled PV+ pyramidal neurons that formed 15% of the neuronal population.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of neurons expressing luteinizing hormone-releasing hormone (LHRH) gene transcripts was mapped in the human hypothalamus and basal forebrain by in situ hybridization and computer-assisted microscopy. Hypothalamic blocks were dissected from five adult males and one adult female and snap frozen in isopentane. The blocks were serially sectioned either in the coronal or in the sagittal plane at a thickness of 20 microns. Approximately every twentieth section was incubated with a 35S-labeled cDNA probe complementary to LHRH mRNA. Specificity was confirmed by hybridization of adjacent sections with a probe targeted to the gonadotropin-associated protein (GAP) region of LHRH messenger ribonucleic acids (mRNA). Maps of neurons containing LHRH mRNA were manually digitized with the aid of an image-combining computer microscope system. We report a much wider distribution and greater numbers of LHRH neurons than have been previously described in the human brain. Three morphological subtypes were observed based on cell size and labeling density: 1) small, heavily labeled, oval or fusiform neurons, located primarily in the medial basal hypothalamus, ventral preoptic area, and periventricular zone; 2) small, oval, sparsely labeled neurons located in the septum and dorsal preoptic region and scattered from the bed nucleus of the stria terminalis to the amygdala ("extended amygdala"); and 3) large round neurons (> 500 microns 2 sectional profile area), intermediate in labeling density, scattered within the magnocellular basal forebrain complex, extended amygdala, ventral pallidum, and putamen. The pronounced differences in morphology, labeling density, and location of the three subtypes suggest that distinct functional subgroups of LHRH neurons exist in the human brain.

A study of the morphology and laminar distribution of nonpyramidal neurons in Golgi-Nissl preparations of electrophysiologically verified auditory cortex was carried out in the adult rabbit. Nonpyramidal neurons were located primarily within laminae I-IV and were only infrequently seen in lamina V and VI. In lamina I, four nonpyramidal cell types were observed: (1) small, spine-free horizontal neurons, (2) small, sparsely spined multipolar neurons with radiate dendrites, (3) large, multipolar neurons with fusiform somata and vertically aligned, sparsely spined dendrites, and (4) small, spine-free neurogliform neurons. The horizontal and small multipolar neurons had tangentially running axons confined to lamina I. The large, fusiform cells had descending axons which arborized in lamina II and occasionally reached lamina III. In lamina II and the upper part of lamina III, seven nonpyramidal cell types were observed: (1) spine-free bipolar neurons with vertically aligned dendrites and axonal arbors; (2) large, (3) medium, and (4) small, spine-free and sparsely spined multipolar neurons, all with locally ramifying axons; (5) pear-shaped cells with highly oriented dendrites which branched toward the pial surface and vertically arborizing axons; (6) multipolar cells with tangentially and vertically oriented dendrites and ascending axons which entered lamina I, and (7) tufted cells with local axons. Three types of nonpyramidal cells were observed in lamina IV and the lower part of lamina III: (1) large, multipolar cells with radiate, spine-free dendrites and stout axons which arborized locally, (2) spiny multipolar cells with vertically aligned dendrites and ascending axons which arborized in lamina II and III via long horizontal collaterals, and (3) spine-free bipolar cells with vertical dendrites and axons which arborized in a narrow vertical column adjacent to the dendrites. Nonpyramidal neurons in lamina V and VI were primarily multipolar cells with sparsely spined and spine-free dendrites. A comparison of these data with those of other species indicates that the neuronal organization of the rabbit auditory cortex is similar to that of the sensory cortex of the rodent but is strikingly different from that of carnivores and primates.

The morphology of lateral preoptic (POL) and lateral hypothalamic (HLA) neurons was studied in 14- to 200-day-old rats with the chlorate-formaldehyde modification of the Golgi method. Drawings of 91 POL and HLA neurons revealed three distinct neuronal types within the MFB based on somatic size and shape and dendritic morphology. Class I neurons, which accounted for 75-80% of the neurons in the MFB, has fusiform or multipolar somata averaging 21 X 14 micron and 2-5 sparsely branched dendrites with a moderate number of sticklike spines. The extensive dendritic domains of Class I neurons ranged from 700 to 1,500 micron and were usually oriented perpendicular to the longitudinal fibers of the MFB. Both nonoriented and oriented Class I neurons were encountered. Nonoriented Class I neurons had expansive dendritic arbors which reached nearly all regions of the MFB in the coronal plane. Oriented Class I neurons had dendritic domains which were confined to specific regions (e.g., ventral-lateral) of the MFB. Class II neurons, which made up approximately 10% of the MFB neurons, had large multipolar somata averaging 30 X 17 micron and 2-5 stout dendrites which were densely covered with hairlike spines. Class II neurons also exhibited spines on their somata and proximal dendritic trunks and had dendritic domains of 700-1,000 micron. Class III neurons had small somata averaging 15 X 12 micron and restricted dendritic arbors of 500-700 micron in diameter. Class III neurons exhibited both spiny and spine-free dendrites and made up 10% of MFB neurons. Because of the parcellation of chemically coded fiber systems within the MFB, individual POL and HLA neurons may not be homogeneous in the type of afferents they receive from other brain areas.

The cytoarchitecture of the prearcuate gyrus, including the region of the physiologically defined frontal eye fields (FEF), was studied in four macaque monkeys (Macaca fascicularis, M. mulatta) to determine if the FEF could be anatomically identified. Brain sections were stained with standard Nissl and, in some cases, myelin stains. Two nonstandard planes of section were used: one tangential to the prearcuate gyrus and the second normal to the most posterior bend of the prearcuate gyrus. The first plane of section was advantageous for studying the location of the FEF with reference to the entire medial-lateral extent of the gyrus and the second allowed good comparisons of the FEF to adjacent anterior and posterior cortical areas. Frontal plane sections through the prearcuate gyrus were also examined in 15 macaque monkeys for comparison with sections cut normal to the posterior bend of the gyrus and tangential to the gyrus. Intracortical microstimulation was performed in three monkeys. The FEF was defined as the area from which low-threshold (less than or equal to 50 microA) saccades could be evoked. The area extended about 10 mm along the anterior bank of the arcuate sulcus. Within the area, saccade amplitudes were represented in a mediolateral, large-to-small topography. No topography of saccade direction was noted within FEF but reversals of saccade direction for any given electrode pass were found. These results confirm the results from our earlier mapping study of FEF (Bruce et al.: J. Neurophysiol. 54:714-734, '85). Cell bodies of large pyramidal cells in layers III and V of the prearcuate gyri from three hemispheres were measured with the aid of an image-combining computer microscope. The distribution of cells of greater than 22 microns diameter or cross-sectional areas of greater than 500 microns 2 were plotted. In one monkey, marker lesions made at microstimulation sites within the FEF or in adjacent non-FEF areas were also plotted. The location of the FEF appeared to coincide with the concentration of large layer V pyramidal cells in the prearcuate gyrus rather than with any previously mapped cytoarchitectonic area. The numbers of large pyramids in layer V were noticeably reduced along the lip of the prearcuate gyrus and at dorsomedial and ventrolateral locations which were outside the physiologically defined FEF.

A study of the morphometry and laminar distribution of spine-free nonpyramidal neurons in electrophysiologically verified primary auditory cortex was carried out in adult rabbits. By using image-combining computer microscopy, the locations of all impregnated neurons in 300-micrometers Golgi-Cox Nissl sections through the auditory cortex were determined. Spine-free non-pyramidal neurons constitute nearly 72% of the nonpyramidal neurons present. They are distributed in a band extending from 450 to 750 micrometers beneath the pial surface corresponding to laminae III and IV. A combination of dendritic stick, Fourier, and statistical analyses revealed a highly significant spatial orientation of their dendrite systems along a vertical axis parallel to the apical dendrites of pyramidal neurons. A significant tangential orientation of dendrites along a dorsal-ventral axis was also found. A radial analysis of the dendrite systems revealed that the pronounced vertical orientation of spine-free nonpyramidal neurons is due to (1) directed dendritic growth along the vertical axis, (2) decreased branching and rapid termination of tangentially oriented dendrites, and (3) increased branching of vertically growing dendrites. The radial analysis also revealed that the longest branches are those directed toward the white matter.

We have studied the postnatal development of lamina III/IV spine-free nonpyramidal neurons in the auditory cortex of the New Zealand white rabbit. The morphology and dendritic branching pattern of single cells impregnated with a Golgi-Cox variant were analyzed with the aid of camera lucida drawings and three-dimensional reconstructions obtained with a computer microscope. Sample sizes of 20 neurons were obtained at birth (day 0), postnatal day (PD) 3, 6, 9, 12, 15, 21, and 30 days of age. Normative data were also available from PD-60 and young adult rabbits studied previously. At birth, lamina II-IV have not yet emerged from the cortical plate; immature nonpyramidal neurons at the bottom of the cortical plate (presumptive layer IV) are characterized by short, vertically oriented dendrites. Growth-cone-like structures are present along the shafts and at the tips of the dendrites. At birth, soma area and total dendritic length are, respectively, 34 and 10% of adult values. The cortical plate acquires a trilaminar appearance at PD-3. The six-layered cortex is present by PD-6. During the first postnatal week dendritic length increases fourfold and is accompanied by a significant increase in both terminal and preterminal dendritic growth cones. At the onset of hearing at PD-6, there is a significant proliferation of dendrites and branches to 144 and 200% of adult levels, respectively. These supernumerary dendrites are rapidly lost during the second postnatal week, at which time the somata and dendrites become covered with spines. The loss of higher-order dendrites occurs more gradually; the number of dendritic branches is still 116% of adult values at PD-30. Spine density peaks between days PD-12 and PD-15, and then gradually diminishes until the cells are sparsely spined or spine free by PD-30. Total dendritic length increases in a linear fashion up to PD-15, at which time it is 80% of adult values. An analysis of terminal and intermediate branches demonstrated that the increase in total dendritic length after PD-6 is due entirely to the growth of terminal dendrites. Total dendritic length attains adult levels by PD-30. Spatial analyses revealed that a vertical orientation of dendrites is present at birth. Associated with the onset of hearing at PD-6, there is an explosive elaboration of dendrites toward the pial surface.(ABSTRACT TRUNCATED AT 400 WORDS)

In order to examine the influence of afferent input on nonpyramidal dendrite development in the auditory cortex, unilateral deafening was carried out in neonatal rabbits at birth, approximately 6 days prior to the onset of hearing. Deafening was produced by surgical removal of the incus and stapes ossicles, aspiration of the cochlear perilymph, and kanamycin injection into the oval window. At 60 days of age, acoustic stimulation of the deafened ear was unable to evoke auditory brainstem responses. The brains of experimental and littermate control rabbits were processed according to the Golgi-Cox Nissl method. The dendritic systems of lamina III/IV spine-free nonpyramidal cells in the auditory cortex contralateral to the deafened ear were digitized from 340-micron-thick coronal sections with the aid of a computer microscope. Three-dimensional spatial and statistical analyses revealed that nonpyramidal dendrite length in neonatally deafened rabbits increased 27% relative to littermate controls. A fan-in projection analysis revealed that the increased dendrite length in the deafened animals was maximum in the tangential direction and toward the white matter. Computer rotation of digitized neurons from neonatally deafened rabbits also revealed evidence of abnormal dendritic growth in the form of recurved dendrites. We interpret our results to indicate that unilateral cochlear destruction early in development causes a reorganization of the ascending auditory pathway which extends to the contralateral cerebral cortex. Because the auditory cortex contralateral to the deafened ear still receives acoustic input from the undamaged ipsilateral ear, normal nonpyramidal dendritic growth in the auditory cortex is, in part, dependent upon afferent activity arising from both ears.