Glutamatergic Neurokinin 3 Receptor Neurons in the Median Preoptic Nucleus Modulate Heat-Defense Pathways in Female Mice.

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.

Ablation of KNDy Neurons Results in Hypogonadotropic Hypogonadism and Amplifies the Steroid-Induced LH Surge in Female Rats.

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 17ß-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 17ß-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.

Neurokinin 3 Receptor-Expressing Neurons in the Median Preoptic Nucleus Modulate Heat-Dissipation Effectors in the Female Rat.

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-17ß (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.

Electrophysiology of arcuate neurokinin B neurons in female Tac2-EGFP transgenic mice.

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 17ß-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.

Developmental changes in the connective tissues of the porcine recurrent laryngeal nerve.

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.

Role for kisspeptin/neurokinin B/dynorphin (KNDy) neurons in cutaneous vasodilatation and the estrogen modulation of body temperature.

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 17ß-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.

Arcuate kisspeptin/neurokinin B/dynorphin (KNDy) neurons mediate the estrogen suppression of gonadotropin secretion and body weight.

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 17ß-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 17ß-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.

Prenatal nicotine exposure alters glycinergic and GABAergic control of respiratory frequency in the neonatal rat brainstem-spinal cord preparation.

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.

Dendritic orientation and laminar architecture in the rabbit auditory thalamus.

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.

Functional subregions in primary auditory cortex defined by thalamocortical terminal arbors: an electrophysiological and anterograde labeling study.

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.

Cell types and response properties of neurons in the ventral division of the medial geniculate body of the rabbit.

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.