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We have proposed that arcuate neurons coexpressing kisspeptin, neurokinin B, and dynorphin (KNDy neurons) contribute to hot flushes via projections to neurokinin 3 receptor (NK3R)-expressing neurons in the median preoptic nucleus (MnPO). To characterize the thermoregulatory role of MnPO NK3R neurons in female mice, we ablated these neurons using injections of saporin toxin conjugated to a selective NK3R agonist. Loss of MnPO NK3R neurons increased the core temperature (TCORE) during the light phase, with the frequency distributions indicating a regulated shift in the balance point. The increase in TCORE in the ablated mice occurred despite changes in the ambient temperature and regardless of estrogen status. We next determined whether an acute increase in ambient temperature or higher TCORE would induce Fos in preoptic enhanced green fluorescent protein (EGFP)-immunoreactive neurons in Tacr3-EGFP mice. Fos activation was increased in the MnPO but no induction of Fos was found in NK3R (EGFP-immunoreactive) neurons. Thus, MnPO NK3R neurons are not activated by warm thermosensors in the skin or viscera and are not warm-sensitive neurons. Finally, RNAscope was used to determine whether Tacr3 (NK3R) mRNA was coexpressed with vesicular glutamate transporter 2 or vesicular γ-aminobutyric acid (GABA) transporter mRNA, markers of glutamatergic and GABAergic neurotransmission, respectively. In the MnPO, 94% of NK3R neurons were glutamatergic, but in the adjacent medial preoptic area, 97% of NK3R neurons were GABAergic. Thus, NK3R neurons in the MnPO are glutamatergic and play a role in reducing TCORE but are not activated by warm thermal stimuli (internal or external). These findings suggest that KNDy neurons modulate thermosensory pathways for heat defense indirectly via a subpopulation of glutamatergic MnPO neurons that express NK3R., (Copyright © 2019 Endocrine Society.)

In the human infundibular (arcuate) nucleus, a subpopulation of neurons coexpress kisspeptin and neurokinin B (NKB), 2 peptides required for normal reproductive function. A homologous group of neurons exists in the arcuate nucleus of rodents, termed KNDy neurons based on the coexpression of kisspeptin, NKB, and dynorphin. To study their function, we recently developed a method to selectively ablate KNDy neurons using NK3-SAP, a neurokinin 3 receptor agonist conjugated to saporin (SAP). Here, we ablated KNDy neurons in female rats to determine whether these neurons are required for estrous cyclicity and the steroid induced LH surge. NK3-SAP or Blank-SAP (control) was microinjected into the arcuate nucleus using stereotaxic surgery. After monitoring vaginal smears for 3-4 weeks, rats were ovariectomized and given 17β-estradiol and progesterone in a regimen that induced an afternoon LH surge. Rats were killed at the time of peak LH levels, and brains were harvested for NKB and dual labeled GnRH/Fos immunohistochemistry. In ovary-intact rats, ablation of KNDy neurons resulted in hypogonadotropic hypogonadism, characterized by low levels of serum LH, constant diestrus, ovarian atrophy with increased follicular atresia, and uterine atrophy. Surprisingly, the 17β-estradiol and progesterone-induced LH surge was 3 times higher in KNDy-ablated rats. Despite the marked increase in the magnitude of the LH surge, the number of GnRH or anterior ventral periventricular nucleus neurons expressing Fos was not significantly different between groups. Our studies show that KNDy neurons are essential for tonic levels of serum LH and estrous cyclicity and may play a role in limiting the magnitude of the LH surge.

KNDy neurons facilitate tail skin vasodilation and modulate the effects of estradiol on thermoregulation. We hypothesize that KNDy neurons influence cutaneous vasodilation via projections to neurons in the median preoptic nucleus (MnPO) that express the neurokinin 3 receptor (NK3R). In support of this hypothesis, focal microinjections of senktide, an NK3R agonist, into the MnPO lowers core temperature (TCORE) in the female rat. To further study the role of MnPO NK3R neurons in thermoregulation, these neurons were specifically ablated using a conjugate of a selective NK3R agonist and saporin (NK3-SAP). NK3-SAP or blank-SAP (control) was injected into the MnPO/medial septum. Tail skin temperature (TSKIN) and TCORE were measured in ovariectomized rats exposed to 3 ambient temperatures (TAMBIENT) before and after estradiol-17β (E2) treatment. Before killing, we injected senktide (sc), monitored TCORE for 70 minutes, and harvested brains for Fos immunohistochemistry. Ablation of MnPO NK3R neurons lowered TSKIN at neutral and subneutral TAMBIENT regardless of E2 treatment. However, ablation did not prevent the effects of E2 on TCORE and TSKIN. In control rats, senktide injections induced hypothermia with numerous Fos-immunoreactive cells in the MnPO. In contrast, in NK3-SAP rats, senktide did not alter TCORE and minimal Fos-immunoreactive neurons were identified in the MnPO. These data show that NK3R neurons in the MnPO are required for the hypothermic effects of senktide but not for the E2 modulation of thermoregulation. The lower TSKIN in NK3-SAP-injected rats suggests that MnPO NK3R neurons, like KNDy neurons, facilitate cutaneous vasodilation, an important heat-dissipation effector.

Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2-enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β-oestradiol (E2)-treated mice were visualised with anti-biotin immunohistochemistry and reconstructed in three dimensions with computer-assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E2 treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin-releasing hormone-immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E2 treatment of OVX Tac2-EGFP mice induces structural changes in the somata and dendrites of KNDy neurones., (© 2015 British Society for Neuroendocrinology.)

Neurons in the arcuate nucleus that coexpress kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) play an important role in the modulation of reproduction by estrogens. Here, we study the anatomical and electrophysiological properties of arcuate NKB neurons in heterozygous female transgenic mice with enhanced green fluorescent protein (EGFP) under the control of the Tac2 (NKB) promoter (Tac2-EGFP mice). The onset of puberty, estrous cyclicity, and serum LH were comparable between Tac2-EGFP and wild-type mice. The location of EGFP-immunoreactive neurons was consistent with previous descriptions of Tac2 mRNA-expressing neurons in the rodent. In the arcuate nucleus, nearly 80% of EGFP neurons expressed pro-NKB-immunoreactivity. Moreover, EGFP fluorescent intensity in arcuate neurons was increased by ovariectomy and reduced by 17β-estradiol (E2) treatment. Electrophysiology of single cells in tissue slices was used to examine the effects of chronic E2 treatment on Tac2-EGFP neurons in the arcuate nucleus of ovariectomized mice. Whole-cell recordings revealed arcuate NKB neurons to be either spontaneously active or silent in both groups. E2 had no significant effect on the basic electrophysiological properties or spontaneous firing frequencies. Arcuate NKB neurons exhibited either tonic or phasic firing patterns in response to a series of square-pulse current injections. Notably, E2 reduced the number of action potentials evoked by depolarizing current injections. This study demonstrates the utility of the Tac2-EGFP mouse for electrophysiological and morphological studies of KNDy neurons in tissue slices. In parallel to E2 negative feedback on LH secretion, E2 decreased the intensity of the EGFP signal and reduced the excitability of NKB neurons in the arcuate nucleus of ovariectomized Tac2-EGFP mice.

The recurrent laryngeal nerve (RLN) branches from the vagus cranial nerve to innervate structures important for voicing and swallowing. Damage to this nerve, commonly associated with surgery or idiopathic etiologies that largely occur with aging, results in impaired voicing and swallowing (Myssiorek, 2004). Sunderland proposed a model of peripheral nerve damage whereby a nerve's ability to resist damage from stretch and compression is determined by the quantity and composition of its epineurial connective tissues (Sunderland, 1951). Thus, it would be expected that epineurium differs depending upon the forces imposed on a nerve within its anatomical setting. The purpose of this study was to investigate RLN epineurium quantity and composition with development. A porcine model (piglet vs. juvenile) was used because of the similarity between porcine and human laryngeal innervation, anatomy and function. The entire RLN was excised bilaterally, and stereological methods were used to quantify the composition of epineurial connective tissues. Compared with the piglet, the juvenile pig RLN was double the diameter. While the piglet had no differences in the percentage of epineurial collagen and adipose between proximal and distal segments of both sides of the RLN, the juvenile pig had a greater percentage of collagen in the proximal segment of both sides of the RLN and a greater percentage of adipose in the distal segment of the left RLN compared with the proximal segment. In addition, unlike the piglet, the juvenile pig had a greater number of fascicles in the proximal than distal segment of the RLN, regardless of nerve side. These findings are consistent with predicted patterns associated with the different anatomical settings of the left and right RLN, show that the RLN changes with age, and support Sunderland's model., (© 2013 Anatomical Society.)

Estrogen withdrawal in menopausal women leads to hot flushes, a syndrome characterized by the episodic activation of heat dissipation effectors. Despite the extraordinary number of individuals affected, the etiology of flushes remains an enigma. Because menopause is accompanied by marked alterations in hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurons, we hypothesized that these neurons could contribute to the generation of flushes. To determine if KNDy neurons participate in the regulation of body temperature, we evaluated the thermoregulatory effects of ablating KNDy neurons by injecting a selective toxin for neurokinin-3 expressing neurons [NK(3)-saporin (SAP)] into the rat arcuate nucleus. Remarkably, KNDy neuron ablation consistently reduced tail-skin temperature (T(SKIN)), indicating that KNDy neurons facilitate cutaneous vasodilatation, an important heat dissipation effector. Moreover, KNDy ablation blocked the reduction of T(SKIN) by 17β-estradiol (E(2)), which occurred in the environmental chamber during the light phase, but did not affect the E(2) suppression of T(SKIN) during the dark phase. At the high ambient temperature of 33 °C, the average core temperature (T(CORE)) of ovariectomized (OVX) control rats was significantly elevated, and this value was reduced by E(2) replacement. In contrast, the average T(CORE) of OVX, KNDy-ablated rats was lower than OVX control rats at 33 °C, and not altered by E(2) replacement. These data provide unique evidence that KNDy neurons promote cutaneous vasodilatation and participate in the E(2) modulation of body temperature. Because cutaneous vasodilatation is a cardinal sign of a hot flush, these results support the hypothesis that KNDy neurons could play a role in the generation of flushes.

Estrogen withdrawal increases gonadotropin secretion and body weight, but the critical cell populations mediating these effects are not well understood. Recent studies have focused on a subpopulation of hypothalamic arcuate neurons that coexpress estrogen receptor α, neurokinin 3 receptor (NK(3)R), kisspeptin, neurokinin B, and dynorphin for the regulation of reproduction. To investigate the function of kisspeptin/neurokinin B/dynorphin (KNDy) neurons, a novel method was developed to ablate these cells using a selective NK(3)R agonist conjugated to the ribosome-inactivating toxin, saporin (NK(3)-SAP). Stereotaxic injections of NK(3)-SAP in the arcuate nucleus ablated KNDy neurons, as demonstrated by the near-complete loss of NK(3)R, NKB, and kisspeptin-immunoreactive (ir) neurons and depletion of the majority of arcuate dynorphin-ir neurons. Selectivity was demonstrated by the preservation of proopiomelanocortin, neuropeptide Y, and GnRH-ir elements in the arcuate nucleus and median eminence. In control rats, ovariectomy (OVX) markedly increased serum LH, FSH, and body weight, and these parameters were subsequently decreased by treatment with 17β-estradiol. KNDy neuron ablation prevented the rise in serum LH after OVX and attenuated the rise in serum FSH. KNDy neuron ablation did not completely block the suppressive effects of E(2) on gonadotropin secretion, a finding consistent with redundant pathways for estrogen negative feedback. However, regardless of estrogen status, KNDy-ablated rats had lower levels of serum gonadotropins compared with controls. Surprisingly, KNDy neuron ablation prevented the dramatic effects of OVX and 17β-estradiol (E(2)) replacement on body weight and abdominal girth. These data provide evidence that arcuate KNDy neurons are essential for tonic gonadotropin secretion, the rise in LH after removal of E(2), and the E(2) modulation of body weight.

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Bath application of GABA-A receptor agonists in neonatal rat brainstem-spinal cord preparations (BSSC) reduces respiratory frequency, an effect that is enhanced by prenatal nicotine exposure. Here we test the hypothesis that these effects can be reproduced by microinjection of GABAergic and glycinergic agonists into the pre-Botzinger complex region (PBC). We recorded the activity of phrenic motor axons from the fourth cervical ventral root in 1-3 days old BSSC that were exposed to either nicotine (6 mg/(kg day)) or saline prenatally. Microinjection of glycine or muscimol into the PBC caused abrupt, reversible apnea in all experiments. Apnea duration with glycine averaged 50.3+/-5 s in saline-exposed (N=12), and 95.7+/-9.9 s in nicotine-exposed (N=12) neonates (P<0.001). Apnea duration with muscimol averaged 51+/-5.1 s in saline-exposed (N=10), and 86+/-10.6 s in nicotine-exposed (N=12) neonates (P<0.05). These data show that prenatal nicotine exposure alters development of central ventilatory control, and that neurons in the PBC region are involved.

A model of auditory thalamic organization is presented incorporating cellular laminae, oriented dendritic arbors and tectothalamic axons as a basis for the tonotopic map at this level of the central auditory system. The heart of this model is the laminar organization of neuronal somata in the ventral division of the medial geniculate body (MGV) of the rabbit, visible in routine Nissl stains. Microelectrode studies have demonstrated a step-wise ascending progression of best frequencies perpendicular to the cell layers. The dendritic arbors of MGV neurons are aligned parallel to the cellular laminae and dendritic tree width along the frequency axis corresponds closely with the frequency steps seen in microelectrode studies. In the laminated subdivision, tectothalamic axons terminate in the form of bands closely aligned with the laminae and dendritic arbors of thalamic relay neurons. The bands of tectothalamic axons extend in the anterior-posterior (A-P) plane forming a dorsal-ventral series of stacked frequency slabs. In the pars ovoidea region, the homologous spiraling of somata, dendritic fields and tectothalamic axons appear to represent a low-frequency area in this species. At least two types of tectothalamic terminals were found within the bands: large boutons frequently arranged in a glomerular pattern and smaller boutons arising from fine caliber axons. We propose that the rabbit is an ideal model to investigate the structural-functional basis of functional maps in the mammalian auditory forebrain.

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.)

Several functional maps have been described in primary auditory cortex, including those related to frequency, tuning, latency, binaurality, and intensity. Many of these maps are arranged in a discontinuous or patchy manner. Similarly, thalamocortical projections arising from the ventral division of the medial geniculate body to the primary auditory cortex are also patchy. We used anterograde labeling and electrophysiological methods to examine the relationship between thalamocortical patches and auditory cortical maps. Biotinylated dextran-amine was deposited into physiologically characterized sites in the ventral division of the medial geniculate body of New Zealand white rabbits. Approximately 7 d later, the animal was again anesthetized and the ipsilateral auditory cortex was mapped with tungsten microelectrodes. Multi-unit physiological data were obtained for the following characteristics: best frequency (BF), binaurality, response type, latency, sharpness of tuning, and threshold. Immunocytochemical methods were used to reveal the injection site in the ventral division of the medial geniculate body as well as the anterogradely labeled thalamocortical afferents in the auditory cortex. In 86% of the cases (12 of 14), entry into a thalamocortical patch was associated with a marked change in physiological responses. A consistent BF and binaural class were usually observed within a patch. The patches appear to innervate distinct functional regions coding frequency and binaurality. A model is presented showing how patchy thalamocortical projections participate in the formation of tonotopic and binaural maps in primary auditory cortex.

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.)

Cellular laminae within the tonotopically organized ventral division of the medial geniculate body (MGV) of the cat have been proposed as the anatomical substrate for physiologically defined isofrequency contours. In most species, the laminae are not visible with routine Nissl stains, but are defined by the dendritic fields of principal cells and the terminal arbors of afferents arising from the inferior colliculus. In the present study, we have used the rabbit to directly examine the relationship between the laminar and tonotopic organization of the MGV. Best frequency maps of the MGV in anesthetized adult New Zealand white rabbits were generated from cluster responses recorded at 30-100 microm intervals to randomly presented tone bursts. Parallel vertical penetrations, roughly perpendicular to the laminae, revealed a low-to-high frequency gradient within the MGV. Non-laminated regions of the ventral division, generally found at the rostral or caudal poles, did not demonstrate a systematic frequency gradient. In contrast to a predicted smooth gradient, best frequencies shifted in discrete steps across the axis of the laminae. A similar step-wise frequency gradient has been shown in the central nucleus of the inferior colliculus of the cat. It is proposed that the central laminated core of the MGV represents an efficient architecture for creating narrow frequency filters involved in fine spectral analysis.

Recent studies have demonstrated that gonadectomy of adult male rats induces dendritic growth of neuroendocrine neurons in the arcuate nucleus. We have hypothesized that these changes are secondary to the loss of testosterone negative feedback. In the present study, we examined the effects of testosterone replacement on the dendritic morphology of arcuate neuroendocrine neurons in castrated rats. Rats were orchidectomized and implanted with silastic capsules designed to produce physiological levels of plasma testosterone (n=9) or empty silastic capsules (n=9) for 2 months. Retrograde labeling with systemically injected Fluoro-Gold, followed by intracellular injection of labeled neurons in a fixed slice preparation, were used to visualize arcuate neuroendocrine neurons. Quantitative analysis of dendritic morphology was performed using three-dimensional computer reconstruction. Serum levels of LH (luteinizing hormone) and testosterone were measured by radioimmunoassay. Treatment of castrated rats with physiological levels of testosterone significantly reduced dendritic length, volume and terminal branch number relative to the castrated rats receiving empty silastic capsules. Dendritic spine density was also greater in the testosterone-treated animals, although the total numbers of spines per dendrite was not significantly different between the two groups. In addition, testosterone replacement was effective in reducing serum LH to levels found in intact rats. These studies demonstrate that testosterone replacement suppresses the dendritic outgrowth of arcuate neuroendocrine neurons that occurs in response to castration. The parallel changes in dendritic arbor and serum LH after castration and hormone replacement suggests that the suppressive effects of testosterone are related to steroid negative feedback.

The injection of anterograde tracers into the ventral division of the medial geniculate body (MGV) of both rats and rabbits labels terminal axons in layer I of auditory cortex as well as the more conventional terminal arbors in layers III/IV. Whether these layer I projections represent a separate lemniscal pathway to the molecular layer or arise as collaterals of axons terminating in III/IV has not been addressed. Focal injections of the anterograde tracers biocytin or biotinylated dextran amine were made into the MGV of young rabbits. Serial section reconstruction of single MGV axons in auditory cortex revealed that layer I axons were collaterals of thalamocortical afferents that formed multiple divergent patches within III/IV. MGV collaterals to layer I often coursed tangentially for several millimeters before terminating. In some cases, the layer I collaterals descended to arborize within a thalamocortical patch in layers III/IV. These results suggest considerable radial and tangential divergence in the auditory thalamocortical pathway and argue for an expanded role for layer I in the processing of specific sensory stimuli., (Copyright 1999 Elsevier Science B.V.)

Human menopause is associated with hypertrophy and increased gene expression of neurokinin (NKB) neurons in the infundibular (arcuate) nucleus of the hypothalamus. We have hypothesized that these changes are secondary to gonadal failure. In the present study, we determined that orchidectomy resulted in an increase in the mean profile area and the number of neurons expressing NKB mRNA in the rat arcuate nucleus. No changes were seen when orchidectomy was combined with testosterone or estradiol replacement. These findings support our hypothesis and demonstrate that gonadal steroids modulate NKB neurons in the arcuate nucleus of adult male rats., (Copyright 1999 Elsevier Science B.V.)

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 expression of parvalbumin-like immunoreactivity (PV-LIR) was examined in the mystacial representation within the primary somatosensory cortex (SI) of postnatal day 21 and adult rabbits. PV-LIR was expressed in a prominent vibrissa-like array of patches in layer IV despite the fact that barrels were indistinct in the cytoarchitecture. Each patch consisted of dense terminal-like PV-LIR and a preferential concentration of intensely labeled stellate neurons. Layer V contained scattered small and large intensely labeled basket cells. Layer Vb had a distinct layer of lightly labeled large pyramidal cells that received labeled basket cell terminations. Upper layer VI also contained patches of terminal-like PV-LIR that were in register with the overlying vibrissae pattern. These patches also contained a preferential distribution of labeled non-pyramidal cells as well as modified pyramidal cells. These results suggest that PV-LIR in rabbits delineates cortical modules composed of thalamocortical afferents and inhibitory local circuits in the absence of a distinct barrel cytoarchitecture. In contrast, prior studies of rat SI cortex have revealed a distinct barrel cytoarchitecture but a uniform distribution of PV-LIR. The differences in PV-LIR between rodents and lagomorphs within the vibrissae representation in SI may be related to species differences in thalamic and local cortical circuits devoted to the whisker sense.

Thalamic afferents originating in the ventral division of the medial geniculate body (vMGB) terminate in patches within lamina III/IV of the primary auditory cortex in adult rabbits. Focal iontophoretic injections of the anterograde tracer, biocytin, were made into the vMGB of neonatal rabbits to examine the morphological organization of auditory thalamocortical (TC) afferents prior to hearing onset. TC afferents terminated in distinct patches as early as postnatal day 1 (PD-0 = day of birth), 6 days before the behavioral onset of hearing. In contrast to TC afferents in adults, the terminal arbors of neonatal vMGB axons occupied the entire depth of the cortical plate and lamina I. Serial section reconstructions revealed that single vMGB axons in neonates branched to form multiple patches within the cortical plate. Collaterals also extended to lamina I where they coursed tangentially for several millimeters. An unusual feature of the neonatal TC patches was the contribution of descending collaterals from axons coursing in lamina I. The presence of distinct patches prior to hearing onset indicates that the segregation of auditory TC axons occurs in the absence of acoustically driven activity. The extensive postnatal remodeling of TC axons, however, may indicate activity-dependent refinement of arbor size.

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.

Thalamocortical afferents to the primary auditory cortex of the rabbit were labeled by the iontophoretic injection of the anterograde tracers PHA-L or biocytin into the ventral division of the medial geniculate body (vMGB). Single injections of either tracer into the vMGB labeled multiple "patches" of afferent axons in lamina III/IV of the ipsilateral auditory cortex. Serial section analysis revealed that single patches were elongated in the rostral-caudal axis forming bands of approximately 2 mm in length. The orientation of the bands was similar to the isofrequency contours of the tonotopic maps derived from prior electrophysiological experiments. Within the coronal plane, the topography of the patches is remarkably similar to the intermittent distribution of binaural interaction subclasses described in physiological studies. Our results are consistent with a model of vMGB organization containing functionally distinct, parallel anatomical pathways to AI.

A striking neuronal hypertrophy occurs in the infundibular nucleus of postmenopausal women. To determine the gender specificity of this response, we measured the areas of neuronal profiles in the infundibular nucleus of young (21, 32, and 41 years) and older (60, 61, and 68 years) men and compared them to data reported previously from the hypothalami of pre-(28, 32, and 40 years) and postmenopausal women (58, 62, and 74 years). Sagittal blocks of formalin-fixed hypothalami were paraffin embedded, serially sectioned and stained with cresyl-violet. The profile areas of 2,429 infundibular neurons were manually digitized using an image-combining computer microscope. The contralateral hypothalamus of each subject was cryoprotected, frozen-sectioned in the coronal plane and also stained with cresyl violet. The infundibular nuclear volume and the total number of neurons were estimated from the coronal sections using stereological methods. The mean profile area of infundibular neurons from older men (176.6 +/- 1.7 microns 2) was significantly larger than that of young men (147.0 +/- 1.3 microns 2). There was also a significant increase in the density of hypertrophied neurons (> 226 microns 2 profile area) in the infundibular nucleus of older men. There was no difference in infundibular nucleus associated with an average neurons was significantly increased in the older men. A comparison with previous data obtained from pre- and postmenopausal women revealed that the profile area of infundibular neurons was equal in young men and young women. However, the profile area of neurons in the postmenopausal women (190.4 +/- 2.1 microns 2) was significantly greater than that of older men.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer microscopy and in situ hybridization were used to investigate neuronal hypertrophy in the infundibular nucleus of postmenopausal women. In the first experiment, hypothalami from premenopausal (n = 3) and postmenopausal (n = 3) women were formalin fixed, paraffin embedded, serially sectioned, and stained with cresyl violet. Soma areas of more than 3500 neurons were digitized using an image-combining computer microscope. The mean cross-sectional area of infundibular neurons in the postmenopausal women was 30% greater than that in premenopausal women, with no change in cell density. The mean cross-sectional area of mammillary neurons was unchanged, indicating that the infundibular neuronal hypertrophy was not an artifact of tissue processing. In the second experiment, hypothalami from premenopausal (n = 3) and postmenopausal (n = 2) women were frozen, serially sectioned, and incubated with a 48-base synthetic cDNA probe complementary to estrogen receptor (ER) mRNA. Adjacent sections were incubated with a cDNA probe complementary to GnRH mRNA. Morphometric analysis revealed that the mean cross-sectional area of infundibular neurons expressing the ER gene in the postmenopausal women was twice as large as the mean area in premenopausal hypothalami. The hypertrophied neurons did not contain GnRH mRNA. Finally, analysis of the infundibular nucleus from an oophorectomized 38-yr-old woman also revealed hypertrophied neurons containing ER mRNA. These data support the hypothesis that hypertrophy of infundibular neurons in postmenopausal women is secondary to loss of the inhibitory feedback of ovarian steroids.

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.

Extracellular single unit recordings were made from units within the lateral preoptic area (LPO) of infant rats from 1 day of age through 31 days of age (postweaning). The rats were anesthetized with dial-urethane and basal (spontaneous) spike rates were recorded for 10--20 min. The rats also received pain-arousal stimulation (tail-pinch and sc injection of formalin solution), and osmotic stimulation (sc or ip injection of hyperosmotic NaCl or sucrose solutions). LPO units were spontaneously active in neonatal rats; however, basal spike rates increased during development. Also increasing during development was the percentage of LPO units which displayed alteration of spike rate in response to hyperosmotic injections (osmosensitive). However, LPO units were found that were responsive to severe pain-arousal stimulation at all ages tested. The developmental changes in LPO unit activity characteristics paralleled the morphological development of LPO neurons and the development of osmotically induced drinking behaviors.

The image-combining computer microscope is a new configuration of light microscope and computer graphics instrumentation which provides remarkable morphometric capabilities over a wide range of applications. The key component is the well known camera lucida, an image-combining device that is used to superimpose the computer graphics image upon the microscope's image of the object under study. Illumination need not be limited to the bright-field variety. The image superposition permits the investigator to study the object and acquire data from it while always looking through the microscope oculars. A computer controlled stepping motor driven stage is integrated with translations of the graphics image to permit image superposition and data acquisition at maximum visual magnification over the full expanse of a 25 X 25 mm tissue section. The investigator controls the system by means of a graphics tablet and a "Menu" area visible to him in the microscope's field of view. Spatial data in all 3 dimensions can be acquired easily since the z-axis is also stepping motor controlled. EM micrograph transparencies can be studied similarly. Stereological applications are possible by superimposing test grids upon the image. The system is self-contained and carries out data analysis programs written in C or Basic. Simplicity of operation has been emphasized throughout.

Single-unit recordings were made from lateral hypothalamic area (LHA) neurons of infant (8 through 21 days of age) male and female albino rats. Basal (or spontaneous) firing rates of LHA neurons were recorded for 20 min from rats 8 through 21 days of age, and mean basal firing rates did not differ across ages. Basal firing rates of LHA neurons in infant rats ranged from less than 1 spike/sec up to 27 spikes/sec, with 56% of the basal rates less than 1 spike/sec. Infant rats also received hypertonic saline injections (and control injections) to determine osmosensitivity characteristics of LHA neurons. Across ages, 56%--88% of LHA neurons were osmosensitive, with the highest percentages of osmosensitive units found in rats 14--21 days of age. Infant rats showed LHA unit electrophysiological characteristics that were quite similar to those obtained from LHA neurons in adult rats with respect to basal firing rates and osmosensitivity, however, LHA neuron activity in infants was not modulated by sensory stimuli as is frequently reported for adults. The results are discussed in terms of functional development of the LHA.

Male and female albino rat pups sustained septal destruction at 10 days of age and body weight and water intakes were measured daily throughout development until 200 days of age. During development the septal and control rats received a battery of drinking tests (e.g., cellular dehydration, hypovolemia, renin). Septal rats (males and females) were hyperdipsic for daily water intakes as early as 31 days of age and the daily hyperdipsia persisted through 200 days of age. On the battery of drinking tests, septal rats consumed water at control volumes on all tests except water deprivation (following which females were hyperdipsic) and food deprivation (during which both males and females were hyperdipsic). Urine output-water intake relationships were determined, and a series of food deprivation tests were conducted during development to determine the etiology of the developmental septal hyperdipsia. All results suggest that hyperdipsia associated with septal destruction during the preweaning period is a primary condition and not secondary to altered output mechanisms, abnormal feeding-drinking patterns, or displacement behavior.

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.

Neonatal rabbits were unilaterally deafened at birth by surgical removal of the stapes, aspiration of the cochlear lymph, and kanamycin injection into the oval window. At 60 days of age, all rabbits were screened with brain stem evoked response tests in order to establish the efficacy of the deafening procedure. The auditory cortex contralateral to the destroyed cochlea was processed according to Golgi-Cox/Nissl procedures. Temporal bone histology revealed nearly complete outer hair cell loss in the damaged cochlea. The dendritic system of lamina III/IV pyramidal neurons contralateral to the deafened ear was digitized from frontal sections using a computer microscope system. Spine counts were also made along the basal dendrites. Spine counts revealed that neonatally deafened rabbits and 38.7% fewer spines along their basal dendrites. No differences between experimental and control rabbits were found in terms of soma cross-sectional area, total number of basal dendrites, total number of dendritic branches and total basal dendritic length. A fan-in projection of the dendritic system revealed no changes in the radial growth of basal dendrites resulting from the early acoustic trauma. In a prior study, spine-free nonpyramidal neurons in the same sections revealed altered dendritic growth and abnormally recurved dendrites. The separate response of pyramidal and nonpyramidal cell types to early cochlear damage is evidence for the different role of epigenetic determinants of dendritic form and orientation in sensory neocortical neurons.

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)

Despite impressive advances in the application of computer image analysis to cytology, many of the identification tasks that cytologists are called on to perform remain refractory to automated image analysis. The major reason is that a large fraction of these images, though simple for a human to deal with, are too complex to yield to current image analysis methodologies. It may be years before automated computer image analysis is reduced to clinical practicality. Even then, it is not clear that all cytologic image analyses will prove amenable to automation. In the meantime, semiautomatic image analysis (computer-aided microscopy) can provide a viable alternative, especially to persistently difficult image analysis problems. In semiautomatic image analysis, the onerous tasks of data acquisition--e.g., stage movement, data entry and storage--are left to the computer, while the decision-making tasks-e.g., identifying a cell's morphologic class--are left to the observer. Such a system proves to be easy and flexible to use as well as economical to build. It can also provide a reliable data base for the later evaluation of fully automated systems as they are developed. One such semiautomatic system, the Image Combining Computer Microscope (ICCM), is described, and the range of its application is illustrated. Some of the examples of ICCM applications discussed are: neuronal cell plots, three-dimensional dendrite tracking, serial section reconstruction of axons and mapping of plaques and tangles in Alzheimer's disease. They illustrate how powerful a semiautomated system can be in handling complex image analysis problems. It is suggested that semiautomated image analysis provides a viable long-range alternative to many cytologic image analysis problems.

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.

Male (N = 9) and female (N = 7) infant albino rats sustained bilateral lateral preoptic area (LPO) destruction on the tenth day of life. During development and as adults, these brain-damaged rats were subjected to repeated testing of drinking behaviors in response to cellular and extracellular dehydration. Immediately following LPO destruction the pups ceased suckling and most rats required tube-feeding to maintain life; however, all pups were maintaining body weight through voluntary suckling and/or feeding-drinking by 20-21 days of age. Daily water intakes were elevated from 30-42 days of age, following which intakes decreased to control vonse to water deprivation, polyethylene glycol injection and food deprivation treatments. These results are consistent with the suggestion that the LPO contains osmoreceptors for drinking behavior, and the present results suggest that such osmoreceptors, may be functional by the tenth day of life for rats.

The fan-in projection is a computer graphical method of projecting onto a half plane the branching patterns of dendrites and axons that have already been 3-dimensionally digitized. It is suited for neurons possessing an axis of orientation (or elongation), such as cortical neurons. This axis is taken as the polar axis of a spherical coordinate system whose center is the soma. For cortical neurons, the equatorial plane corresponds to the tangential plane. Co-latitude is measured with respect to the positive polar axis. Longitude is discarded. What results is a projection in which dendrites and axons appear to be growing in a half plane whose boundary is the polar axis. The projection eliminates many of the distorting effects of depth foreshortening seen in conventional projections. In so doing it helps one to visualize branching properties that would otherwise be obscure.