Neurogliaform cells: neurochemistry, spatial arrangement, and their role in the neocortical inhibitory system

The neurogliaform cells (NGFC) localized in area 4 of the human motor cortex were found to express choline acetyl transferase (ChAT), GABA, and calbindin. ChAT-positive neurons were located in cortical layer II. Their dendrites lay in a close proximity to those of pyramid cells, neighbouring neurogliaform cells, and bodies and dendrites of other cortical neurons. The NGFCs revealed by Golgi staining fell into two groups. Cells of the first group had locally (within cortical layer II) spreading axons, while those of the second group had axons extending into the adjacent layers. Neurochemical heterogeneity of NGFCs is discussed in the context of information processing in cortical modules and interaction of excitatory and inhibitory interneurons.     

Neocortical Axon Arbors Trade-off Material and Conduction Delay Conservation

The brain contains a complex network of axons rapidly communicating information between billions of synaptically connected neurons. The morphology of individual axons, therefore, defines the course of information flow within the brain. More than a century ago, Ramón y Cajal proposed that conservation laws to save material (wire) length and limit conduction delay regulate the design of individual axon arbors in cerebral cortex. Yet the spatial and temporal communication costs of single neocortical axons remain undefined. Here, using reconstructions of in vivo labelled excitatory spiny cell and inhibitory basket cell intracortical axons combined with a variety of graph optimization algorithms, we empirically investigated Cajal''s conservation laws in cerebral cortex for whole three-dimensional (3D) axon arbors, to our knowledge the first study of its kind. We found intracortical axons were significantly longer than optimal. The temporal cost of cortical axons was also suboptimal though far superior to wire-minimized arbors. We discovered that cortical axon branching appears to promote a low temporal dispersion of axonal latencies and a tight relationship between cortical distance and axonal latency. In addition, inhibitory basket cell axonal latencies may occur within a much narrower temporal window than excitatory spiny cell axons, which may help boost signal detection. Thus, to optimize neuronal network communication we find that a modest excess of axonal wire is traded-off to enhance arbor temporal economy and precision. Our results offer insight into the principles of brain organization and communication in and development of grey matter, where temporal precision is a crucial prerequisite for coincidence detection, synchronization and rapid network oscillations.     

Synaptic input to the axon hillock and initial segment of inhibitory interneurons in the cerebellar cortex of the rat

Summary The axon hillock (AH) and initial segment (IS) of 10 Golgi neurons and 6 basket cells in the cerebellar cortex of the rat were investigated by electron microscopy using serial sections. An average of 10.4 and 11.3 synaptic terminals were observed to establish synaptic contact with the axon hillock region of Golgi and basket cells, respectively. Most of these terminals were identified as the varicosities of the ascending parallel fibers. It is suggested that the focal innervation of AH regions represents an excitatory input pattern which is basically different from the randomly distributed, huge, parallel-fiber input onto the dendritic trees of Golgi and basket cells. In contrast to Golgi and basket neurons, no accumulation of parallel-fiber synapses was observed around the AH of stellate cells. The IS proper of the three neuronal types were devoid of true axo-axonal synapses.     

Nerve growth factor-induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF-induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP-43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF-induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75NTR expression. We propose that p75NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF.     

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.     

Studies on the neurosecretory system and retrocerebral endocrine glands of Nezara viridula Linn. (Heteroptera: Pentatomidae)

The neurosecretory system and retrocerebral endocrine glands of Nezara viridula Linn. have been described on the basis of in situ preparations and histological sections employing the paraldehyde fuchsin (PF) and performic acid-victoria blue (PAVB) techniques. In the brain of N. viridula, there are two medial groups–each consisting of five neurosecretory cells which belong to A-type. The lateral neurosecretory cells are absent. The axons of the two groups of medial neurosecretory cells (MNC) compose the two bundles of neurosecretory pathways (NSP) that decussate in the anterodorsal part of the protocerebrum. The two pathways, after the cross-over, run deep into the protocerebrum and deutocerebrum and emerge as NCC-I from the tritocerebrum. The nervi corporis cardiaci-I (NCC-I) of each side which are heavily loaded with NSM terminate in the aorta wall. Thus, the neurosecretory material (NSM), elaborated in the medial neurosecretory cells of the brain, is stored in the aortic wall and nervi corporis cardiaci-I (NCC-I). The NCC-II are very short nerves that originate from the tritocerebrum and terminate in the corpora cardiaca (CC) of their side. Below the aorta, but dorsal to the oesophagus, lie two oval or spherical corpora cardiaca. A corpus allatum (CA) lies posterior to the corpora cardiaca (CC). The corpora cardiaca do not contain NSM; only the intrinsic secretion of their cells has been occasionally observed which stains orange or green with PF staining method. The corpus allatum sometimes exhibits PF positive granules of cerebral origin. A new connection between the corpus allatum and aorta has been recorded. The suboesophageal ganglion contains two neurosecretory cells of A-type which, in structure and staining behaviour, are similar to the medial neurosecretory cells of the brain. The course and termination of axons of suboesophageal ganglion neurosecretory cells, and the storage organ for the secretion of these cells have been reported. It is suggested that the aortic wall and NCC-I axons function as neurohaemal organ for cerebral and suboesophageal secretions.     

Neurons and their interconnections in the frontal cortex of monkeys

It was shown by the Golgi and Golgi-Kopsch method that pyramidal cells of layers II–IV in the frontal cortex of the monkeyMacaca rhesus have numeruous, mainly recurrent axon collaterals by means of which they form vertical connections. Pyramidal cells with ascending axons are found. Axons of stellate basket neurons unite pyramidal cells in both horizontal (modules) and vertical (micromodules) directions; depending on the direction of the axon collaterals, two groups of stellate neurons can be distinguished. Groups of 14 to 16 pyramidal cells whose apical dendrites are connected into bundles were found. Axons of pyramidal cells in layers II–IV descend in the composition of the pyramidal tract and give off collaterals which run toward the bodies and dendrites of neighboring pyramidal cells, united into the same group, forming terminal and en passant junctions. Besides bundles, special kinds of "local" cell groups with U-shaped axons are found.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 15, No. 2, pp. 115–120, March–April, 1983.     

Extrastriate feedback to primary visual cortex in primates: a quantitative analysis of connectivity.

Knowledge-based or top-down influences on primary visual cortex (area V1) are believed to originate from information conveyed by extrastriate feedback axon connections. Understanding how this information is communicated to area V1 neurons relies in part on elucidating the quantitative as well as the qualitative nature of extrastriate pathway connectivity. A quantitative analysis of the connectivity based on anatomical data regarding the feedback pathway from extrastriate area V2 to area V1 in macaque monkey suggests (i) a total of around ten million or more area V2 axons project to area V1; (ii) the mean number of synaptic inputs from area V2 per upper-layer pyramidal cell in area V1 is less than 6% of all excitatory inputs; and (iii) the mean degree of convergence of area V2 afferents may be high, perhaps more than 100 afferent axons per cell. These results are consistent with empirical observations of the density of radial myelinated axons present in the upper layers in macaque area V1 and the proportion of excitatory extrastriate feedback synaptic inputs onto upper-layer neurons in rat visual cortex. Thus, in primate area V1, extrastriate feedback synapses onto upper-layer cells may, like geniculocortical afferent synapses onto layer IVC neurons, form only a small percentage of the total excitatory synaptic input.     

Nerve growth factor–induced growth of sympathetic axons into the optic tract of mature mice is enhanced by an absence of p75NTR expression

Postganglionic sympathetic axons display a remarkable ability for new collateral growth in response to local increases in nerve growth factor (NGF). Elevating NGF levels within the brain also induces the directional growth of sympathetic axons, but not within myelinated pathways of adult mammals. In this investigation, we provide in vivo evidence that sympathetic axons are capable of NGF‐induced collateral growth through the microenvironment of mature myelinated pathways, especially in the absence of the p75 neurotrophin receptor (NTR). In transgenic mice overexpressing NGF centrally and expressing p75^NTR, only a few varicose sympathetic axons invade the optic tract after the first month of postnatal life. In other transgenic mice overexpressing NGF centrally but lacking p75^NTR expression, the incidence of sympathetic axons within this myelinated tract substantially increases. Moreover, numerous unmyelinated sympathetic axons cluster together to form large processes extending through the optic tract; such structures are first seen 8 weeks after birth. Only these large axon bundles display prominent immunostaining for GAP‐43, which is preferentially localized to the sympathetic fibers, since nonmyelinating Schwann cells are not associated with these axon bundles. These data provide the first direct evidence that sympathetic axons are indeed capable of NGF‐induced collateral growth into myelinated tracts of mature mammals, and that their continued growth through this microenvironment is markedly enhanced by the absence of p75^NTR expression. We propose that p75^NTR among sympathetic axons may either directly or indirectly limit collateral branching of these fibers in response to increased levels of NGF. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 51–66, 1999     

Efficacy of thalamocortical and intracortical synaptic connections: quanta, innervation, and reliability.

Thalamocortical (TC) synapses carry information into the neocortex, but they are far outnumbered by excitatory intracortical (IC) synapses. We measured the synaptic properties that determine the efficacy of TC and IC axons converging onto spiny neurons of layer 4 in the mouse somatosensory cortex. Quantal events from TC and IC synapses were indistinguishable. However, TC axons had, on average, about 3 times more release sites than IC axons, and the mean release probability at TC synapses was about 1.5 times higher than that at IC synapses. Differences of innervation ratio and release probability make the average TC connection several times more effective than the average IC connection, and may allow small numbers of TC axons to dominate the activity of cortical layer 4 cells during sensory inflow.     

Parallel driving and modulatory pathways link the prefrontal cortex and thalamus

Pathways linking the thalamus and cortex mediate our daily shifts from states of attention to quiet rest, or sleep, yet little is known about their architecture in high-order neural systems associated with cognition, emotion and action. We provide novel evidence for neurochemical and synaptic specificity of two complementary circuits linking one such system, the prefrontal cortex with the ventral anterior thalamic nucleus in primates. One circuit originated from the neurochemical group of parvalbumin-positive thalamic neurons and projected focally through large terminals to the middle cortical layers, resembling 'drivers' in sensory pathways. Parvalbumin thalamic neurons, in turn, were innervated by small 'modulatory' type cortical terminals, forming asymmetric (presumed excitatory) synapses at thalamic sites enriched with the specialized metabotropic glutamate receptors. A second circuit had a complementary organization: it originated from the neurochemical group of calbindin-positive thalamic neurons and terminated through small 'modulatory' terminals over long distances in the superficial prefrontal layers. Calbindin thalamic neurons, in turn, were innervated by prefrontal axons through small and large terminals that formed asymmetric synapses preferentially at sites with ionotropic glutamate receptors, consistent with a driving pathway. The largely parallel thalamo-cortical pathways terminated among distinct and laminar-specific neurochemical classes of inhibitory neurons that differ markedly in inhibitory control. The balance of activation of these parallel circuits that link a high-order association cortex with the thalamus may allow shifts to different states of consciousness, in processes that are disrupted in psychiatric diseases.     

Environmental control of collateral branching and target invasion of mitral cell axons during development

During development, mitral cell axons, the major efferents of the olfactory bulb, exhibit a protracted waiting period in the lateral olfactory tract (LOT) before giving off collateral branches and innervating the target olfactory cortex. To investigate the target invasion mechanism, a series of heterochronic and heterotopic cocultures of olfactory bulbs with various olfactory cortical strips were conducted. These experiments indicated that development of collateral branches is triggered by environmental cues but not by intrinsic mechanisms in mitral cells. The collateral-inducing cues are apparently different from the cues directing outgrowth of primary mitral cell axons. Coculture experiments also indicated that the target olfactory cortex undergoes a developmental change to become accessible to mitral cell fibers. Primary mitral cell axons, however, still preferred the LOT position over such accessible piriform cortex when encountered both the locations. These results suggest that mitral cell projection comprises multiple steps which are controlled by various environmental cues.     

How Informative Are Spatial CA3 Representations Established by the Dentate Gyrus?

In the mammalian hippocampus, the dentate gyrus (DG) is characterized by sparse and powerful unidirectional projections to CA3 pyramidal cells, the so-called mossy fibers. Mossy fiber synapses appear to duplicate, in terms of the information they convey, what CA3 cells already receive from entorhinal cortex layer II cells, which project both to the dentate gyrus and to CA3. Computational models of episodic memory have hypothesized that the function of the mossy fibers is to enforce a new, well separated pattern of activity onto CA3 cells, to represent a new memory, prevailing over the interference produced by the traces of older memories already stored on CA3 recurrent collateral connections. Can this hypothesis apply also to spatial representations, as described by recent neurophysiological recordings in rats? To address this issue quantitatively, we estimate the amount of information DG can impart on a new CA3 pattern of spatial activity, using both mathematical analysis and computer simulations of a simplified model. We confirm that, also in the spatial case, the observed sparse connectivity and level of activity are most appropriate for driving memory storage – and not to initiate retrieval. Surprisingly, the model also indicates that even when DG codes just for space, much of the information it passes on to CA3 acquires a non-spatial and episodic character, akin to that of a random number generator. It is suggested that further hippocampal processing is required to make full spatial use of DG inputs.     

mGluRs Modulate Strength and Timing of Excitatory Transmission in Hippocampal Area CA3

Excitatory transmission within hippocampal area CA3 stems from three major glutamatergic pathways: the perforant path formed by axons of layer II stellate cells in the entorhinal cortex, the mossy fiber axons originating from the dentate gyrus granule cells, and the recurrent axon collaterals of CA3 pyramidal cells. The synaptic communication of each of these pathways is modulated by metabotropic glutamate receptors that fine-tune the signal by affecting both the timing and strength of the connection. Within area CA3 of the hippocampus, group I mGluRs (mGluR1 and mGluR5) are expressed postsynaptically, whereas group II (mGluR2 and mGluR3) and III mGluRs (mGluR4, mGluR7, and mGluR8) are expressed presynaptically. Receptors from each group have been demonstrated to be required for different forms of pre- and postsynaptic long-term plasticity and also have been implicated in regulating short-term plasticity. A recent observation has demonstrated that a presynaptically expressed mGluR can affect the timing of action potentials elicited in the postsynaptic target. Interestingly, mGluRs can be distributed in a target-specific manner, such that synaptic input from one presynaptic neuron can be modulated by different receptors at each of its postsynaptic targets. Consequently, mGluRs provide a mechanism for synaptic specialization of glutamatergic transmission in the hippocampus. This review will highlight the variability in mGluR modulation of excitatory transmission within area CA3 with an emphasis on how these receptors contribute to the strength and timing of network activity within pyramidal cells and interneurons.     

Central afferent pathways of long hair sensilla in the ventral nerve cord of the Indian black scorpion,Heterometrus fulvipes Koch

Histological and electrophysiological studies of identified long hair sensilla (LHS) have provided information on primary afferent fibre pathways in the ventral nerve cord of the Indian black scorpion, Heterometrus fulvipes.Cobalt-filling of single LHS on the metasoma showed that sensory axons enter the respective segmental ganglion, ascend ipsilaterally through the next anterior ganglia and terminate in a 4th ganglion. In each ganglion, these plurisegmental fibres give off collateral branches that terminate in the ganglionic neuropil. Fibres entering heterolateral connectives were not found.Recordings from peripheral nerves after deflections of a hair showed single or multiple spike discharges. A single spike could be recorded from ipsilateral anterior connectives of the ventral nerve cord, indicating a through-conductance of the sensory pathways. Strong deflections of a single hair activated several ipsilateral and fewer contralateral ascending interneurons and some segmentai motor neurons. Behavioral studies demonstrate the mechanoreceptive function of the LHS.The present study provides evidence in support of the notion that sensory afferents of the postabdomen in the scorpion bring about rapid, co-ordinated intersegmental movements of the multisegmented tail of the scorpion.Abbreviations CNS central nervous system - LHS long hair sensillum - TR trichobothria     

Cerebellar localization and colocalization of GABA and calcium binding protein-D28K

Immunocytochemical studies using antibodies raised against the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA) and against the 28 Kd vitamin D dependent calcium binding protein (calbindin) in the cerebellum, are reviewed. The GABA immunoreactive neurones found in the cerebellar cortex were the Purkinje cell (PC), the three classes of intrinsic inhibitory interneurones, stellate, basket and Golgi cells and the cells of Lugaro. Some of the neurons of the cerebellar nuclei were also found to be GABA immunoreactive. A part of these could be identified as extrinsic neurones projecting either back to the cerebellar cortex, or to the inferior olive, both these pathways being topographically highly organized but arising from independent parent neurons. The presumed inhibitory function of these two pathways are discussed. Calbindin immunoreactivity in the cerebellum was confined to the PCs, staining concerned the whole cell including soma, branching dendrites, axons and axons terminals. The antibody, which appears to be tightly bound to the PC in vivo, failed to stain some of the PC when cerebellar slices maintained in vitro were studied. The stability of the antigen-antibody binding and the use of calbindin as a marker specific for the PC in the cerebellum, is discussed. Co-localization of GABA with calbindin as well as with other calcium binding proteins are reported to be found in the PCs. While these co-localizations have led to much speculation, conclusive functional roles for them have not been identified at present.     

Network properties of memory trace formation in the hippocampus.

Based on the experimental evidence from his laboratory and the relevant literature the Author outlines a formal model of memory trace formation. During exploratory (theta) behaviors the neocortical information is transmitted to the hippocampus via the fast-firing granule cells which may induce a weak and transient heterosynaptic potentiation in a subgroup of CA3 pyramidal cells. The weakly potentiated CA3 neurons will then initiate population bursts upon the termination of exploratory activity (sharp wave state). It is assumed that recurrent excitation during the population burst is strongest on those cells which initiated the population event. It is suggested that the strong excitatory drive brought about by the sharp wave-concurrent population bursts during consummatory behaviors, immobility, and slow wave sleep may be sufficient for the induction of long-term synaptic modification in the initiator neurons of the CA3 region and in their targets in CA1. In this two-stage model both exploratory (theta) and sharp wave states of the hippocampus are essential and any interference that might modify the structure of the population bursts (e.g., epileptic spikes) are detrimental to memory trace formation.     

Spinal processing: anatomy and physiology of spinal nociceptive mechanisms

The processing of nociceptive input that occurs at the spinal level represents the first stage of effective control over its access to higher regions of the central nervous system. Recent developments in both the anatomy and physiology of nociceptive processing pathways at this level are beginning to yield an integrated understanding of structure and function. Most small afferent axons terminate in the more superficial laminae of dorsal horn, but technical difficulties have, until recently, prevented analysis of the functional properties of identified small fibres. A direct input of nociceptive afferents on to particular dorsal horn neurons is difficult to establish in view of the slow impulse conduction in these fibres and the small size of target neurons in the substantia gelatinosa. The small cells themselves are being analysed for relations between structure and function, using physiological, intracellular staining and immunocytochemical techniques to characterize their properties. They appear to be a highly heterogeneous population with many sub-classes, whether typed according to the transmitter they contain, e.g. enkephalin, to their physiological responses: whether excitatory or inhibitory to nociceptive and other inputs, or to both. The multireceptive neurons that project out of the dorsal horn toward supraspinal regions are, in general, located in deeper laminae and are likely to receive nociceptive information through polysynaptic pathways. The nocireceptive neurons in lamina I, which receive exclusively nociceptive inputs from myelinated and non-myelinated afferents project, at least in part, to thalamic and brain stem regions. Polysynaptic nociceptive pathways in dorsal horn may be subject to different controls from neurons in laminae I and II. Tonic descending inhibition is operative on the former and it is becoming clearly established that descending systems such as those containing noradrenaline, can regulate the access of nociceptive information to higher levels. The mechanisms of such descending controls and the importance of their interaction with segmental control systems, such as those involving the dynorphin opioids, are just beginning to be understood. Many somatosensory neurons in dorsal horn, both the large cells, some of which project supraspinally, and the small cells of superficial laminae, receive convergent nociceptive and non-nociceptive inputs. Although solely nociresponsive neurons are clearly likely to fill a role in the processing and signalling of pain in the conscious central nervous system, the way in which such useful specificity could be conveyed by multireceptive neurons is difficult to appreciate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cerebellar Cortex Granular Layer Interneurons in the Macaque Monkey Are Functionally Driven by Mossy Fiber Pathways through Net Excitation or Inhibition

The granular layer is the input layer of the cerebellar cortex. It receives information through mossy fibers, which contact local granular layer interneurons (GLIs) and granular layer output neurons (granule cells). GLIs provide one of the first signal processing stages in the cerebellar cortex by exciting or inhibiting granule cells. Despite the importance of this early processing stage for later cerebellar computations, the responses of GLIs and the functional connections of mossy fibers with GLIs in awake animals are poorly understood. Here, we recorded GLIs and mossy fibers in the macaque ventral-paraflocculus (VPFL) during oculomotor tasks, providing the first full inventory of GLI responses in the VPFL of awake primates. We found that while mossy fiber responses are characterized by a linear monotonic relationship between firing rate and eye position, GLIs show complex response profiles characterized by “eye position fields” and single or double directional tunings. For the majority of GLIs, prominent features of their responses can be explained by assuming that a single GLI receives inputs from mossy fibers with similar or opposite directional preferences, and that these mossy fiber inputs influence GLI discharge through net excitatory or inhibitory pathways. Importantly, GLIs receiving mossy fiber inputs through these putative excitatory and inhibitory pathways show different firing properties, suggesting that they indeed correspond to two distinct classes of interneurons. We propose a new interpretation of the information flow through the cerebellar cortex granular layer, in which mossy fiber input patterns drive the responses of GLIs not only through excitatory but also through net inhibitory pathways, and that excited and inhibited GLIs can be identified based on their responses and their intrinsic properties.     

FMRFamide-like immunoreactivity in the ventral ganglion of the fly Sarcophaga bullata: metamorphic changes.

1. Localization of FMRFamide-like immunoreactivity was examined in the ventral ganglion of the fly Sarcophaga bullata using the indirect immunofluorescent method. 2. There are six large cells in the thoracic ganglion which are highly immunoreactive at all stages of development. 3. During metamorphosis the thoracic FLI neurons shift their position from ventrolateral to mid-ventral position and their axons terminate and elaborate a highly immunoreactive dorsal neural sheath. 4. It is suggested that the dorsal neural sheath may function as a neurohaemal organ from which FMRFamide-like substances may be released into the haemolymph to act as neurohormones.