Total Neuronal Population Research Articles

Network Effects of Glutamate on Neuronal Activity in the Medial Septum/Diagonal Band Complex In Vitro

Inter-neuronal interactions within the medial septum/diagonal band complex (MSDB) are of great interest as this region is believed to be the hippocampal theta rhythm pacemaker. However, the role of glutamatergic system in functioning of the septal cells is yet unclear. Here, we demonstrate for the first time the effects of glutamate in physiological concentration (1 microM) on the MSDB neuronal spontaneous and evoked activities in vitro. These effects (activation of 70% and inhibition of 30% of responsive neurons) differed in pacemaker and non-pacemaker cells. Pacemaker cells were always activated under glutamate, whereas non-pacemaker neurons could be either activated or inhibited. Indeed, in the burst pacemakers, glutamate increased the frequency of rhythmic activity. In a total MSDB neuron population, in 30% of neurons glutamate applications modified responses to the electrical stimulation by unifying the temporal parameters of neuron responses. Along with the increase in the theta-burst frequency, this indicates that the glutamatergic system is involved in the process ofintraseptal synchronization. Obtained data shed light on the role ofglutamatergic system in septal neuron interactions and broaden our understanding of theta oscillation mechanisms in the septo-hippocampal system.

The percentage of interneurons in the dorsal striatum of the rat, cat, monkey and human: A critique of the evidence

In the published literature on mammals, there are various answers to the question: What percentage of the total population of neurons within the dorsal striatum is represented by interneurons? The percentage of interneurons is reported to be 26% for the human, 23% or 4–6% for the monkey, 5–6% or <3% for the rodent, 4–5% for the cat and 5–10% for the mammal. Based on a critique of the literature, including the use of stereological approaches, caution is needed when citing historical non-stereological data on the percentage of interneurons in the dorsal striatum of the cat, monkey and human. In these species, the vast majority of neurons in the dorsal striatum are spiny projection neurons and a smaller number are interneurons, yet the precise percentage is currently unknown. Hence, in the absence of stereological data on the absolute number of all neuronal subtypes for the cat, monkey and human, it is currently unknown if there is a species difference, or not, for the percentage of interneurons in the dorsal striatum of the human and monkey versus the cat and rat. Modern stereological data indicate that the percentage of interneurons in the rat dorsal striatum is <3%. More specifically, in the Sprague-Dawley rat, 0.8% of all neurons are somatostatin/neuropeptide Y/GABA interneurons, 0.4% are cholinergic interneurons, ∼0.6% are GABA/parvalbumin interneurons and 0.5% are GABA/calretinin interneurons. Current stereological data also indicate that the percentage of interneurons in the dorsal striatum is <1% for cholinergic interneurons in the Sprague-Dawley rat and human.

Food cues and ghrelin recruit the same neuronal circuitry

Cues that are associated with the availability of food are known to trigger food anticipatory activity (FAA). This activity is expressed as increased locomotor activity and enables an animal to prepare for maximal utilization of nutritional resources. Although the exact neural network that mediates FAA is still unknown, several studies have revealed that the medial hypothalamus is involved. Interestingly, this area is responsive to the anorexigenic hormone leptin and the orexigenic hormone ghrelin that have been shown to modulate FAA. However, how FAA is regulated by neuronal activity and how leptin and ghrelin modulate this activity is still poorly understood. We aimed to examine how the total neuronal population and individual neurons in the medial hypothalamus respond to cue-signaled food availability in awake, behaving rats. In addition, ghrelin and leptin were injected to investigate whether these hormones could have a modulatory role in the regulation of FAA. Using in vivo electrophysiology, neuronal activity was recorded in the medial hypothalamus in freely moving rats kept on a random feeding schedule, in which a light cue signaled upcoming food delivery. Ghrelin and leptin were administered systemically following the behavioral paradigm. The food-predictive cue induced FAA as well as a significant increase in neural activity on a population level. More importantly, a sub-population of medial hypothalamic neurons displayed highly correlated identical responses to both ghrelin and FAA, suggesting that these neurons are part of the network that regulates FAA. This study reveals a role for ghrelin, but not leptin, signaling within medial hypothalamus in FAA on both a population level and in single cells, identifying a subset of neurons onto which cue information and ghrelin signaling converge, possibly to drive FAA.

Neuropeptide Y‐immunoreactive Neurons in the Cerebral Cortex of Humans and Other Haplorrhine Primates

We examined the distribution of neurons immunoreactive for neuropeptide Y (NPY) in the posterior part of the superior temporal cortex (Brodmann's area 22 or area Tpt) of humans and nonhuman haplorrhine primates. NPY has been implicated in learning and memory and the density of NPY-expressing cortical neurons and axons is reduced in depression, bipolar disorder, schizophrenia, and Alzheimer's disease. Due to the role that NPY plays in both cognition and neurodegenerative diseases, we tested the hypothesis that the density of cortical and interstitial neurons expressing NPY was increased in humans relative to other primate species. The study sample included great apes (chimpanzee and gorilla), Old World monkeys (pigtailed macaque, moor macaque, and baboon) and New World monkeys (squirrel monkey and capuchin). Stereologic methods were used to estimate the density of NPY-immunoreactive (-ir) neurons in layers I-VI of area Tpt and the subjacent white matter. Adjacent Nissl-stained sections were used to calculate local densities of all neurons. The ratio of NPY-ir neurons to total neurons within area Tpt and the total density of NPY-ir neurons within the white matter were compared among species. Overall, NPY-ir neurons represented only an average of 0.006% of the total neuron population. While there were significant differences among species, phylogenetic trends in NPY-ir neuron distributions were not observed and humans did not differ from other primates. However, variation among species warrants further investigation into the distribution of this neuromodulator system.

Intrinsic Innervation of the Persian Squirrel (Sciurus anomalus) Ileum

Most investigations related to the characterisation of the enteric nervous system (ENS) are pivoted on the intestine of small rodents, but few studies are available on the ENS of wild or 'unconventional' rodents. Anti-PGP 9.5 and anti-Hu antibodies were utilised to recognise the distribution pattern of neuronal cell bodies and fibres of the ileum of the Persian squirrel (Sciurus anomalus) ENS. The percentages of subclasses of enteric neurones in the total neuronal population were investigated by neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP), substance P (SP), and calbindin (CALB). Myenteric plexus (MP) and submucosal plexus (SMP) neurones showing nNOS immunoreactivity (IR) were 41±4% and 11±6%, respectively, whereas cells expressing ChAT-IR were 56±9% and 74±16%, respectively. nNOS-IR was co-expressed by 21±2% and 9±4% of the MP and SMP cholinergic neurones, respectively, whereas the nNOS-IR MP and SMP neurones co-expressing ChAT-IR were 86±6% and 89±2%, respectively. CGRP-IR and SP-IR were expressed, respectively, by 13±5% and 6±3% of MP and 18±2% and 2±2% of SMP neurones. CALB-IR was expressed by 22±8% and 56±14% of MP and SMP neurones, respectively. MP and SMP cholinergic neurones co-expressed nNOS-IR (21±2% and 9±4%, respectively) and a very high percentage of nNOS-IR neurones showed ChAT-IR (86±6% and 89±2%, respectively). MP and SMP CALB-IR neurones co-expressed ChAT-IR (100% and 63±11%, respectively) and CGRP-IR (89±5% and 26±7%, respectively). Our data might contribute to the neuroanatomical knowledge of the gastrointestinal tract in exotic mammals and provide a comparison with the available data on other mammals.

Characterization of the myenteric neuronal population and subpopulation of the duodenum of adult wistar rat fed with hypoproteic chow

The effects of severe protein malnutrition (4%) on myenteric neurons of Wistar rat duodenum, in relation to a standard 22%-protein diet for rodents, were assessed in this study. Segments of the duodenum from 10 rats from each nutritional group were submitted to the elaboration of whole mounts - 5 stained with Giemsa to determine the total population of myenteric neurons and the others stained by a histochemical method to detect nervous cells through the NADPH-diaphorase enzyme activity for studying the subpopulation of nitrergic neurons. The area of 100 neurons per animal, totalizing 2,000 neurons, were randomly measured by using the Image Pro-Plus(®)software. Malnourished rats presented 34.38% lower body weight and 10.60% duodenum length reduction when compared to the control group. Quantitative analysis demonstrated no significant differences between control and malnourished group by using Giemsa; however, as the organ reduction was not followed by an increase inversely proportional to the density of neurons, the condition imposed suggests the loss of neurons from the total population. Nevertheless, through NADPH-d histochemistry, there was a neuronal density increase for the malnourished group. There was no significant difference between the groups for both techniques with respect to the morphometric analysis of the body cell.

Cortical inhibitory neuron basket cells: from circuit function to disruption

Despite representing only ∼10–15% of the total cortical and hippocampal neuronal population, local circuit GABAergic inhibitory interneurons represent one of the most diverse cell types in the mammalian central nervous system. In the hippocampal formation alone upwards of 20 distinct cell types have been described, with the number rising each year (Freund & Buzsaki, 1996; Klausberger & Somogyi, 2008; Tricoire et al. 2011). The pressure to diversify is undoubtedly a consequence of the myriad roles played by local circuit interneurons in precisely shaping and modulating features of both local and global circuits. Indeed precision in the circuit largely relies on the laminar positioning of their somato-dendritic axis to receive appropriate afferent input combined with their axonal output, which targets both principal cell and interneuron subcellular compartments.

GnRH Neurons Directly Listen to the Periphery

GnRH Neurons Directly Listen to the Periphery

A Morphometric Comparative Study of the Medial Geniculate Body of the Rabbit and the Fox

Unbiased stereological methods were used to morphometrically examine and compare the medial geniculate body (MGB) of two species from different mammalian orders. The MGB had a similar nuclear pattern, and it was parcelled into three major cytoarchitectural areas: the dorsal nucleus (MGd), the ventral nucleus (MGv) and the medial nucleus (MGm). The MGd was predominant in the fox, where it contributed nearly 50% to the total MGB volume, while in the rabbit, the MGv was insignificantly larger than the MGd. In both species, the percentage contribution of the MGm was the lowest. The MGd in the fox was also characterized by twice as many neurons per mm(3) as in the rabbit, whereas a reverse proportion was observed in the MGm, although the numerical density in the MGv was very similar in both species. The total number of MGB neurons in the fox was over twice higher than that in the rabbit. The variability in the percentage contribution of the MGd, MGv and MGm cells to the total neuronal population of the MGB was different in both mammals. In the rabbit, there was a larger contribution from the MGv and MGm, while in the fox, the MGd was predominant. These data demonstrate that the main areas of the MGB complex differ in terms of the morphometric characteristics in both species. Our results also show that the negative correlation between the volume and numerical density in the sensory centres of the brain might not be as distinct as in the non-sensory brain structures.

Presence and neurochemical coding of intrinsic VIP/PACAP‐positive enteric neurons in the adult zebrafish

In a previous study, detecting the co‐expression of neurochemical markers, five different neuronal classes were identified in the zebrafish enteric nervous system. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) did not yield immunoreactivity (IR) in neuronal cell bodies, only in nerve fibers. At present, we attempted using an in vitro colchicine pretreatment prior to fixation to detect VIP and PACAP in neuronal cell bodies to determine peptidergic subpopulations in the ENS of the adult zebrafish. The proportional distribution of VIP and PACAP was studied using multiple immunofluorescence. Co‐expression with other neurochemical markers was examined. Treatment of colchicine enhanced IR for VIP and PACAP as such that IR was not only observed in neuronal varicosities, but also in neuronal cell bodies. A complete colocalization for VIP and PACAP was observed. VIP/PACAP‐expressing neurons accounted for ±20% of the total neuronal population. Coexpression of VIP/PACAP was observed with calretinin and calbindin (±20%), nNOS (±70%) and serotonin (±20%). ChAT‐IR was not observed after pretreatment with colchicine. These results complement and extend previous data regarding neurochemical properties of the zebrafish ENS. Further immunostainings are necessary to establish which of the subpopulations previous identified coexpress VIP and PACAP.

Enteric neuropathy evoked by repeated cisplatin in the rat

Acute administration of the antitumoral drug cisplatin can induce nausea/emesis and diarrhea. The long-term effects of cisplatin on gastrointestinal motility, particularly after repeated administration, are not well known. Because cisplatin is highly neurotoxic, myenteric neurons can be affected. Our aim was to study the prolonged effects of repeated cisplatin administration in a rat model, focusing on gastrointestinal motor function and myenteric neurons. Rats received saline or cisplatin (1 or 3 mg kg(-1), i.p.) once weekly for 5 weeks. One week after treatment, both upper gastrointestinal transit and colonic activity were evaluated, and tissue samples from ileum, colon and rectum were processed for histological analysis. Intestinal transit was measured invasively (charcoal method). Colonic activity was determined electromyographically. The gut wall structure was evaluated in sections using conventional histology and immunohistochemistry. Whole-mount preparations from the distal colon were labeled for different markers, including nitric oxide synthase (NOS) and calcitonin-gene related peptide (CGRP) to determine relative proportions of myenteric neurons vs the total neuronal population labeled with HuC/D. One week after repeated cisplatin exposure, the upper gastrointestinal transit rate and colonic activity were dose-dependently reduced. The number of NSE- or HuC/D-immunoreactive myenteric neurons per ganglion was decreased; the proportion of CGRP-immunoreactive neurons was decreased, whereas that of NOS-immunoreactive cells was increased. Chronic cisplatin may induce an enteric neuropathy characterized by changes in myenteric neurons associated with marked gastrointestinal motor dysfunction.

The disruption of the ventricular (VZ) and subventricular (SVZ) zones of the ganglionic eminences in hydrocephalic hyh mice is associated to abnormalities in the cortical GABAergic neurons

and intracortical distribution were recorded for the GABAergic neurons. Results and conclusions The mutant hyh mice were characterized by (i) a marked reduction in the width of the cerebral cortex; (ii) a reduction in the total number of GABAergic neurons; (iii) a reduction in the relative number of GABAergic neurons with respect to the total population of neurons (GABA/NeuE); (iv) an abnormal distribution of GABAergic neurons in the cortex layers, with a significant reduction in layers II and III and an increase in layer IV y V. The hyh mutation is associated with: (i) a decreased number of GABAergic neurons migrating from the ganglionic eminences; (ii) abnormal migration of GABAergic neurons through the developing brain cortex; (iii) in hyh mice disruption of the VZ and SVZ is associated to the onset of hydrocephalus and abnormal corticogenesis.

Number estimates of neuronal phenotypes in layer II of the medial entorhinal cortex of rat and mouse

Modelling entorhinal function or evaluating the consequences of neuronal losses which accompany neurodegenerative disorders requires detailed information on the quantitative cellular composition of the normal entorhinal cortex. Using design-based stereological methods, we estimated the numbers, proportions, densities and sectional areas of layer II cells in the medial entorhinal area (MEA), and its constituent caudal entorhinal (CE) and medial entorhinal (ME) fields, in the rat and mouse. We estimated layer II of the MEA to contain ∼58,000 neurons in the rat and ∼24,000 neurons in the mouse. Field CE accounted for more than three-quarters of the total neuron population in both species. In the rat, layer II of the MEA is comprised of 38% ovoid stellate cells, 29% polygonal stellate cells and 17% pyramidal cells. The remainder is comprised of much smaller populations of horizontal bipolar, tripolar, oblique pyramidal and small round cells. In the mouse, MEA layer II is comprised of 52% ovoid stellate cells, 22% polygonal stellate cells and 14% pyramidal cells. Significant species differences in the proportions of ovoid and polygonal stellate cells suggest differences in physiological and functional properties. The majority of MEA layer II cells contribute to the entorhinal-hippocampal pathways. The degree of divergence from MEA layer II cells to the dentate granule cells was similar in the rat and mouse. In both rat and mouse, the only dorsoventral difference we observed is a gradient in polygonal stellate cell sectional area, which may relate to the dorsoventral increase in the size and spacing of individual neuronal firing fields. In summary, we found species-specific cellular compositions of MEA layer II, while, within a species, quantitative parameters other than cell size are stable along the dorsoventral and mediolateral axis of the MEA.

Neuroprotective effects of erythropoietin on 6-hydroxydopamine-treated ventral mesencephalic dopamine-rich cultures

Parkinson's disease (PD) is a neurodegenerative disorder with motor symptoms caused by the loss of dopaminergic (DA) cells and consequently dopamine release in the nigrostriatal system. In vivo and in vitro 6-hydroxydopamine (6-OHDA) PD models are widely used to study the effect of striatal dopamine depletion as well as novel neuroprotective or restorative therapeutic strategies for PD. In the present study, we investigated in vitro the toxicity of 6-OHDA on DA neurons derived from E14 rat ventral mesencephalon (VM) and the neuroprotective efficiency of erythropoietin (Epo) on VM-derived cell cultures against 6-OHDA toxicity. Using E14 VM-derived DA-rich primary cultures, we could demonstrate that 6-OHDA toxicity works in a time-and concentration-dependent way, and leads to cell death not only in DA cells but also in non-DA cells in direct relation to concentration and incubation times. In addition, we found that 6-OHDA toxicity induces caspase-3 activation and an increment of intracellular reactive oxygen species (ROS) in VM-derived cultures. When 6-OHDA-treated VMs were cultured in the presence of the anti-apoptotic protein erythropoietin (Epo), the total neuronal population, including the DA neurons, was protected. However, untreated VM cultures exposed to Epo showed an increase in the total neuronal population, but not an additional increase in DA neuron cell number. These findings suggest that 6-OHDA toxicity is time and concentration-dependent and does not exclusively affect DA neurons. In high concentration and long incubation times, 6-OHDA influences the survival of other neuronal and non-neuronal cell populations derived from the VM cultures. 6-OHDA toxicity induces caspase-3 activation, indicating cell death via the apoptotic pathway which could be restricted or even prevented by pre-exposure to Epo, known to interact via the apoptotic pathway. Our results support and expand on previous findings showing that Epo is an interesting candidate molecule to mediate neuroprotective effects on DA neurons in PD. Furthermore, it could be used in promoting the survival of DA neurons after transplantation in clinical trials.

Cell‐specific expression of neuropeptide Y Y1 receptor immunoreactivity in the rat basolateral amygdala

Activation of neuropeptide Y (NPY) Y1 receptors (Y1r) in the rat basolateral nuclear complex of the amygdala (BLA) produces anxiolysis and interferes with the generation of conditioned fear. NPY is important in regulating the output of the BLA, yet the cell types involved in mediating this response are currently unknown. The current studies employed multiple label immunocytochemistry to determine the distribution of Y1r-immunoreactivity (-ir) in glutamatergic pyramidal and GABAergic cell populations in the BLA using scanning laser confocal stereology. Pyramidal neurons were identified by expression of calcium-calmodulin dependent kinase II (CaMKII-ir) and functionally distinct interneuron subpopulations were distinguished by peptide (cholecystokinin, somatostatin) or calcium-binding protein (parvalbumin, calretinin) content. Throughout the BLA, Y1r-ir was predominately on soma with negligible fiber staining. The high degree of coexpression of Y1r-ir (99.9%) in CaMKII-ir cells suggests that these receptors colocalize on pyramidal cells and that NPY could influence BLA output by directly regulating the activity of these projection neurons. Additionally, Y1r-ir was also colocalized with the interneuronal markers studied. Parvalbumin-ir interneurons, which participate in feedforward inhibition of BLA pyramidal cells, represented the largest number of Y1r expressing interneurons in the BLA ( approximately 4% of the total neuronal population). The anatomical localization of NPY receptors on different cell populations within the BLA provides a testable circuit whereby NPY could modulate the activity of the BLA via actions on both projection cells and interneuronal cell populations.

Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder

The neurochemical phenotypes of the transient receptor potential melastatin-8 (TRPM8)-immunoreactive afferent neurons innervating the rat urinary bladder were examined by using a highly sensitive tyramide signal amplification method, combined with wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing. TRPM8-immunoreactivity was detected in a small proportion of the WGA-HRP-labeled bladder afferent neurons in the dorsal root ganglia of the Th13-L1 (1.14%) and the L6-S1 (1.27%), and these neurons were small in size (<600 μm 2). The 82.6 ± 3.8% of the TRPM8-immunoreactive bladder afferent neurons and 80.9 ± 1.5% of the total population of the TRPM8-immunoreactive afferent neurons in the observed dorsal root ganglia expressed NF200. On the other hand, the proportions of the co-expression of TRPM8 and nociceptive markers such as calcitonin gene-related peptide (CGRP), transient receptor potential vanilloid-1 (TRPV1), and isolectin B4 (IB4) in the bladder afferent neurons (81.5 ± 5.2% for CGRP, 36.1 ± 4.0% for TRPV1, and 15.8 ± 5.5% for IB4) were higher in comparison to those in the total population of the TRPM8-immunoreactive afferent neurons (21.9 ± 2.4% for CGRP, 16.6 ± 1.7% for TRPV1, and 5.4 ± 0.5% for IB4), although no significant difference existed for IB4. Our results suggest that the TRPM8-expressing bladder afferents should be classified as Aδ-fibers and C-fibers, while some of these afferents may be involved in nociceptive sensations.

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

Non‐neuronal BDNF, a key player in development of central sensitization and neuropathic pain

Non‐neuronal BDNF, a key player in development of central sensitization and neuropathic pain

Intrinsic innervation of the horse ileum

This paper describes the morphology and distribution of the enteric nervous system (ENS) cells and fibres immunoreactive for choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), substance P (SP), calcitonin gene-related peptide (CGRP), NF200kDa (NF200), and S100 protein. The percentages of subclasses of enteric neurons in the total neuronal population were investigated by the use of anti-PGP 9.5 or anti-NSE antibodies.ChAT-IR myenteric plexus (MP) and submucosal plexus (SMP) neurons were 66±7% and 74±15%, respectively, whereas those cells expressing nNOS-IR were 38±7% and 5±1%, respectively. MP and SMP neurons expressing both phenotypes were also present. SP-IR was expressed by 14±13% of MP and 66±8% of SMP neurons whereas CGRP-IR was observed only in the SMP (43±6%). NF200-IR was expressed by 61±15% and 91±10% of the MP and SMP neurons, respectively. The majority of the CGRP-IR SMP neurons expressed also SP-IR. Almost all SP-IR neurons in both the plexuses were cholinergic. The present study quantifies the main neuronal subpopulations of the ENS of the horse ileum; these data might be utilized to understand the neuronal modifications which occur in several gastrointestinal tract disorders.

Inhibition of gonadotropin secretion by gonadotropin‐inhibitory hormone: it's all in your head

The seminal work of Geoffery Harris cemented neural substances of hypothalamic origin as key in the control of gonadal function (Harris, 1950). Subsequently, Amoss et al. (1971) and Schally et al. (1971) independently isolated the neuropeptide that directly simulates pituitary gonadotropin secretion and thus, it was termed gonadotropin releasing-hormone (GnRH). Since that time, the search has been underway to identify the location (Wintermantel et al. 2006) and phenotype of neurotransmitters and neuropeptides which allow the GnRH-containing neurons to respond to the multiple internal and external cues (Herbison, 2006) regulating GnRH secretion and by extension controlling sexual reproduction. Tsutsui et al. (2000) provided a new perspective on the control of gonadotropin secretion when they reported a hypothalamic peptide whose inhibitory action on gonadotropin release from quail anterior pituitaries as indicated in figure 1, was consistent with it being a gonadotropin inhibitory hormone. The neuropeptide was termed GnIH for its direct but converse effect (from GnRH) on pituitary gonadotropin secretion (fig. 1). Figure 1 GnIH directly inhibits the activity of GnRH neurons rendering it a putative GnRH-IH in rodents Until most recently, GnIH remained a pituitary GnIH as evidence for hypothalamic site of action was lacking. Now, the groups of Allan Herbison (Ducret et al. 2009) and in this issue of The Journal of Physiology Meenakshi Alreja (Wu et al. 2009) have independently shown that GnIH has direct inhibitory actions on GnRH neurons. The GnIH reduces rates of firing and induces hyperpolarization in substantial portions of the total population of GnRH neurons. Moreover, since GnIH neurons in rats are not neurosecretory (Rizwan et al. 2009), it appears at least in rodents that the GnIH in other vertebrates, may have largely a hypothalamic site of action on gonadotropin secretion (fig. 1). Therefore, the long repudiated GnRH-IH neuropeptide seems to be GnIH. The question remains as to which of the multiple internal and external cues that inhibit activity of GnRH neurons are mediated by activation of GnIH-containing neurons. For example, pulsatile GnRH release is regulated by a negative feedback system. Since the GnRH neurons themselves do not contain classical steroid receptors, gonadal steroids must act through alternative mechanisms such as direct membrane effects or steroid-sensitive presynaptic inputs to GnRH neurons (see Kelly & Ronnekleiv 2008 for review). If GnIH neurons are steroid sensitive, then they could play a pivotal role in inhibition of GnRH secretion via gonadal steroids. Likewise, stress inhibits reproduction. Preliminary evidence suggests that GnIH expression increases in rats during exposure to acute stressors (Kirby et al. 2007). Perhaps the most intriguing aspect of the GnIH peptide is its impact on reproductive behaviour. In male rats, GnIH-related peptides reduce the full sequela of male sexual behaviours (Johnson et al. 2007). Accordingly, input to GnRH neurons from GnIH-containing neurons may provide the link between reproductive hormone secretion and reproductive behaviour (Coquelin & Bronson, 1980). The above speculation notwithstanding, the neurons containing GnIH are poised to exert control of gonadotropin secretion from within the hypothalamus. Thus, the GnIH peptide is likely to emerge as a key regulator of GnRH secretion through its direct actions on GnRH neurons.