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PubMed1986

Substance P- and vasoactive intestinal peptide-containing fibers reach the ovary by independent routes.

The immature rat ovary is innervated by both substance P (SP)- and vasoactive intestinal peptide (VIP)-containing nerve fibers. To determine the origin of these fibers, juvenile rats were sham operated or subjected to transection of the abdominal vagus nerves, superior ovarian nerves, or ovarian plexus nerves or to concomitant transection of the vagal and superior ovarian nerves. Specific immunofluorescent staining of ovarian sections for SP and VIP revealed that VIP innervation to the ovary occurs only via the superior ovarian nerves, whereas ovarian SP innervation is exclusively via the plexus nerves. Thus, specific SP and VIP nerves reach the gland via anatomically different routes. The results indicate that autonomic regulation of ovarian function may be exerted via independent peptidergic neural pathways.

Animals↗

PubMed1998

Trigeminal-reticular connections: possible pathways for nociception-induced cardiovascular reflex responses in the rat.

Cardiovascular regulatory neurons of the ventral medulla and pons are thought to have an important role in the mediation of trigeminal nociception-induced reflex cardiovascular responses. However, the neural pathways that link the spinal trigeminal nucleus with ventral medullary and pontine autonomic cell groups are poorly understood. The present study utilized injections of the highly sensitive anterograde tracer substance biotinylated dextran combined with immunocytochemistry for tyrosine hydroxylase, the synthesizing enzyme for catecholamines, to investigate the distribution and morphology of projections from the spinal trigeminal subnucleus caudalis to ventral medullary and pontine catecholaminergic cell groups. Injection of biotylinated dextran into the dorsal subnucleus caudalis produced dense anterograde labeling in dorsal regions of the medullary and pontine reticular formation including the dorsal medullary reticular field, the parvicellular reticular field, and the parvicellular reticular field pars anterior. In the ventral medullary and pontine reticular formation, light anterograde labeling tended to be distributed in close proximity to the distal dendrites of catecholaminergic neurons located in the C1, A1, and A5 regions. Injections of anterograde tracer into the dorsal medullary reticular field produced dense anterograde labeling in the ventral medullary and pontine reticular formation. Numerous terminal-like varicosities were observed in close proximity to catecholaminergic neurons located in the C1, A1, and A5 regions. These data suggest that trigeminal pain-induced reflex cardiovascular responses involve indirect projections that terminate in the dorsal medullary and pontine reticular formation before reaching ventral medullary and pontine catecholaminergic cell groups known to be involved in cardiovascular regulation.

Animals↗

PubMed1986

Cerebrovascular changes elicited by electrical stimulation of the centromedian-parafascicular complex in rat.

Electrical stimulation of the centromedian-parafascicular complex (CM-Pf) in anesthetized (chloralose) and paralyzed (tubocurarine) rats elicits a widespread cerebrovascular dilatation. Regional cerebral blood flow (rCBF) was measured in dissected tissue samples of 10 brain regions (medulla, pons, cerebellum, inferior colliculus, superior colliculus, frontal parietal and occipital cortices, caudate-putamen and corpus callosum) by [14C]iodoantipyrine method. In unstimulated and sham-operated rats rCBF ranged from 40 +/- 3 (ml/100 g/min) in corpus callosum to 86 +/- 6 (ml/100 g/min) in inferior colliculus. During CM-Pf stimulation, rCBF increased significantly (P less than 0.05, analysis of variance and Scheffe's test) in all cerebral regions bilaterally ranging from +118% in parietal cortex to +38% in cerebellum. Although cerebral vasodilation elicited by CM-Pf stimulation persisted after unilateral transection of the cervical sympathetic trunk, the cortical CBF was significantly reduced (P less than 0.05) on the denervated side. Acute adrenalectomy significantly (P less than 0.05) decreased elevated rCBF during CM-Pf stimulation in all cortical regions (frontal-36%, parietal -34%, and occipital -27%) and in caudate nucleus (-37%). Thus, excitation of neurons originating in, or fibers passing through the CM-Pf can elicit a powerful cerebral vasodilation. The cerebral vasodilation is modulated by cervical sympathectomy and circulating adrenal hormones. We conclude that CM-Pf elicited vasodilation is at least partly mediated by intrinsic neural pathways.

Adrenalectomy↗

PubMed1999

Subcortical neural circuits for ocular accommodation and vergence in primates.

Our current knowledge of the neural bases of vergence and accommodation has increased significantly over the past few years. The behavior of medial rectus motoneurons during vergence, which has been reported by a number of investigators, is described. The behavior of Edinger-Westphal neurons during accommodation is also described, as are the characteristics of midbrain near-response neurons in the supraoculomotor area. Evidence that some of these near-response neurons provide the vergence input to medial rectus motoneurons and possibly the accommodation input to Edinger-Westphal neurons is reviewed. Anatomical studies have shown that the midbrain near-response region receives input from two deep cerebellar nuclei, the posterior interposed and the fastigial nucleus. Single-unit recording in the posterior interposed nucleus has revealed cells that increase their activity during the far-response, and the behavior of these neurons is reviewed. In addition, studies of a precerebellar nucleus, the nucleus reticularis tegmenti pontis, have revealed some cells that increase their activity during the near-response and others that do so during the far-response. The behavior of these neurons is reviewed. This review documents the great strides that are occurring in our understanding of the anatomy and physiology of the neural pathways controlling vergence and accommodation in the primate.

Accommodation, Ocular↗

PubMed2001

Jaw-muscle spindle afferent pathways to the trigeminal motor nucleus in the rat.

Neural pathways conveying proprioceptive feedback from the jaw muscles were studied in rats by combining retrograde and intracellular neuronal labeling. Initially, horseradish peroxidase was iontophoresed unilaterally into the trigeminal motor nucleus (Vmo). Two days later, 1-5 jaw-muscle spindle afferent axons located in the mesencephalic trigeminal nucleus were physiologically identified and intracellularly stained with biotinamide. Stained mesencephalic trigeminal jaw-muscle spindle afferent axon collaterals and boutons were predominantly distributed in the supratrigeminal region (Vsup), Vmo, dorsomedial trigeminal principal sensory nucleus (Vpdm), parvicellular reticular formation (PCRt), alpha division of the parvicellular reticular formation (PCRtA), and dorsomedial portions of the spinal trigeminal subnuclei oralis (Vodm), and interpolaris (Vidm). Numerous neurons retrogradely labeled with horseradish peroxidase from the trigeminal motor nucleus were found bilaterally in the PCRt, PCRtA, Vodm, and Vidm. Retrogradely labeled neurons were also present contralaterally in the Vsup, Vpdm, Vmo, peritrigeminal zone, and bilaterally in the dorsal medullary reticular field. Putative contacts between intracellularly stained mesencephalic trigeminal jaw-muscle spindle afferent boutons and trigeminal premotor neurons retrogradely labeled with horseradish peroxidase were found in the ipsilateral Vodm, PCRtA, and PCRt, as well as the contralateral Vsup, Vmo, Vodm, PCRt, and PCRtA. Thus, multiple disynaptic jaw-muscle spindle afferent-motoneuron circuits exist. These pathways are likely to convey long-latency jaw-muscle stretch reflexes and may contribute to stiffness regulation of the masticatory muscles.

Afferent Pathways↗

PubMed1995

Feedback circuitry within a song-learning pathway.

The song system of birds consists of several neural pathways. One of these, the anterior forebrain pathway, is necessary for the acquisition but not for the production of learned song in zebra finches. It has been shown that the anterior forebrain pathway sequentially connects the following nuclei: the high vocal center, area X of lobus parolfactorius, the medial portion of the dorsolateral thalamic nucleus, the lateral magnocellular nucleus of anterior neostriatum (IMAN), and the robust nucleus of the archistriatum (RA). We now show in zebra finches (Taeniopygia guttata) that IMAN cells that project to RA also project to area X, forming a feedback loop within the anterior forebrain pathway. The axonal endings of the IMAN projection into area X form cohesive and distinct domains. Small injections of tracer in subregions of area X backfill a spatially restricted subset of cells in IMAN, that, in turn, send projections to RA that are arranged in horizontal layers, which may correspond to the functional representation of vocal tract muscles demonstrated by others. We infer from our data that there is a myotopic representation throughout the anterior forebrain pathway. In addition, we suggest that the parcellation of area X into smaller domains by the projection from IMAN highlights a functional architecture within X, which might correspond to units of motor control, to the representation of acoustic features of song, or both.

Animals↗

PubMed1979

Transmitter-specific retrograde labeling in the striato-nigral and raphe-nigral pathways.

Injecting radioactive transmitters into the rat substantia nigra led to retrograde neuronal labeling either in the dorsal raphe nucleus, after 3H-labeled serotonin injection, or in the caudoputamen, after 3H-labeled gamma-aminobutyric acid injection. This differential labeling in projections whose transmitter has been established provides the basis for a histochemical tracing method indicating both connectivity and transmitter specificity of neural pathways.

Animals↗

PubMed2006

Molecular physiology of the neural circuit for calcineurin-dependent associative learning in Caenorhabditis elegans.

How learning and memory is controlled at the neural circuit level is a fundamental question in neuroscience. However, molecular and cellular dissection of the neural circuits underlying learning and memory is extremely complicated in higher animals. Here, we report a simple neural circuit for learning behavior in Caenorhabditis elegans, where the calcium-activated phosphatase, calcineurin, acts as an essential modulator. The calcineurin mutant tax-6 showed defective feeding state-dependent learning behavior for temperature and salt. Surprisingly, defective associative learning between temperature and feeding state was caused by malfunctions of two pairs of directly connected interneurons, AIZ and RIA, in the mature nervous system. Monitoring temperature-evoked Ca2+ concentration changes in the AIZ-RIA neural pathway revealed that starvation, a conditioning factor, downregulated AIZ activity through calcineurin during associative learning between temperature and feeding state. Our results demonstrate the molecular and physiological mechanisms of a simple neural circuit for calcineurin-mediated associative learning behavior.

Animals↗

PubMed1990

Dissociation by chloralose of the cardiovascular and cerebrovascular responses evoked from the cerebellar fastigial nucleus.

We studied the effects of chloralose anesthesia on the elevation in arterial pressure (AP), heart rate (HR), and regional CBF (rCBF) elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthetized with an initial dose of chloralose (40 mg/kg s.c.), paralyzed, and artificially ventilated. The FN was stimulated (50-100 microA, 50 Hz, 1 s on/1 s off) with microelectrodes stereotaxically implanted. During the stimulation AP was carefully maintained within cerebrovascular autoregulation. CBF was measured by the [14C]iodoantipyrine technique with regional dissection. In rats that received only the initial dose of chloralose, FN stimulation elevated rCBF in brain and spinal cord, up to 209 +/- 13% of control in frontal cortex (n = 5; p less than 0.01, analysis of variance). Administration of additional chloralose (10 mg/kg i.v., 30 min prior to measurement of CBF) did not affect resting rCBF (n = 5), the EEG, or the elevation in AP and HR elicited by FN stimulation (n = 4). However, the additional chloralose abolished the elevations in rCBF (n = 5; p greater than 0.05). Thus, the cerebrovasodilation elicited from the FN is more susceptible to the effects of additional anesthesia than the elevation in AP and HR. These results indicate that the cerebrovascular and cardiovascular responses elicited from the FN are functionally distinct and provide additional evidence for the notion that these responses are mediated by different neural pathways and transmitters.

Animals↗

PubMed2006

Noradrenergic inputs to the bed nucleus of the stria terminalis and paraventricular nucleus of the hypothalamus underlie hypothalamic-pituitary-adrenal axis but not hypophagic or conditioned avoidance responses to systemic yohimbine.

The alpha2 adrenoceptor antagonist yohimbine (YO) increases transmitter release from adrenergic/noradrenergic (NA) neurons. Systemic YO activates the hypothalamic-pituitary-adrenal (HPA) axis, inhibits feeding, and supports conditioned flavor avoidance (CFA) in rats. To determine whether these effects require NA inputs to the bed nucleus of the stria terminalis (BNST), vehicle or saporin toxin conjugated to an antibody against dopamine beta hydroxylase (DSAP) was microinjected bilaterally into the BNST to remove its NA inputs. Subsequent tests failed to reveal any lesion effect on the ability of YO (5.0 mg/kg, i.p.) to inhibit food intake or to support CFA. Conversely, HPA axis responses to YO were significantly blunted in DSAP rats. In a terminal experiment, DSAP and control rats were perfused 90-120 min after intraperitoneal injection of YO or vehicle. Brains were processed to reveal Fos immunolabeling and lesion extent. NA fibers were markedly depleted in the BNST and medial parvocellular paraventricular hypothalamus (PVNmp) in DSAP rats, evidence for collateralized NA inputs to these regions. DSAP rats displayed significant loss of caudal medullary NA neurons, and markedly blunted Fos activation in the BNST and in corticotropin-releasing hormone-positive PVNmp neurons after YO. We conclude that a population of medullary NA neurons provides collateral inputs to the BNST and PVNmp, and that these inputs contribute importantly to Fos expression and HPA axis activation after YO treatment. Conversely, NA-mediated activation of BNST and PVNmp neurons is unnecessary for YO to inhibit food intake or support CFA, evidence for the sufficiency of other intact neural pathways in mediating those effects.

Animals↗

PubMed1989

Neural substrates for reflex salivation induced by taste, mechanical, and thermal stimulation of the oral region in decerebrate rats.

In order to investigate the neural mechanisms of reflex salivary secretion, experiments were carried out on anesthetized, decerebrate rats from which the volumes of submandibular salivary secretion and the efferent discharges in the preganglionic parasymapathetic fibers innervating the submandibular gland were recorded. Salivary secretion was induced by either infusing a taste solution, or an aliquot of hot water (45-55 degrees C) into the oral cavity, or by pinching the frontal parts of the oral region with a pair of forceps. The reflex salivation induced by noxious thermal and mechanical stimuli was markedly reduced by lesioning either the caudal (VC), or the interpolar (VI) trigeminal sensory nuclei. Taste-elicited salivary secretion was significantly reduced by lesioning the nucleus of the tractus solitarius (NTS). Of 43 preganglionic parasympathetic fibers sampled, 27 responded to both noxious mechanical and thermal stimulation of the oral region, and to electrical stimulation of the VC. Ten fibers responded only to taste stimulation and to electrical stimulation of the NTS. The remaining 6 fibers responded to both taste and noxious thermal stimulation of the oral region. These fibers responded well to NTS stimulation, but gave only a slight response to VC stimulation. These results suggest that two distinct neural pathways exist which mediate reflex salivation in the lower brain stem of the rat, i.e., the taste pathway via the NTS and the nociceptive pathway via the trigeminal sensory nuclei.

Animals↗

PubMed2005

Recovery of impaired songs following unilateral but not bilateral lesions of nucleus uvaeformis of adult zebra finches.

Zebra finches utilize neural circuits in both cerebral hemispheres to produce their learned songs. Although direct reciprocal connections do not exist between song control nuclei across hemispheres, premotor activity in these nuclei during singing is precisely and continuously coordinated between the hemispheres. We hypothesized that this interhemispheric coordination is mediated by bilateral feedback projections from medullary and midbrain song control nuclei to the thalamic song control nucleus uvaeformis (Uva). Consistent with our hypothesis, bilateral lesions of Uva severely impaired singing. This impairment was long-lasting, as it persisted for at least 35 days after the lesions. Unilateral lesions of Uva on either side also resulted in an immediate singing impairment. However, song recovered substantially after less than 15 days, suggesting a possible compensation by the unlesioned side. Although the acoustic structure of individual syllables recovered fully after unilateral lesioning, subtle changes in the sequencing of syllables were observed after song recovery, suggesting that the lesion led to an alteration in the functioning of the remaining song control network. These results demonstrate that the adult songbird brain can adjust to damage to certain parts of the song control network and recover from their associated singing deficits. The well-identified and localized central neural pathways mediating birdsong production provide an advantageous model system to analyze systematically the sensorimotor contexts and the specific sites and mechanisms for behavioral recovery following partial damage to a behavior-producing neural circuit.

Animals↗

PubMed1991

Non-adrenergic, non-cholinergic neural control of the airways.

1. In addition to the classical cholinergic bronchoconstrictor and adrenergic bronchodilator neural mechanisms, there is a large volume of evidence to suggest the existence of neural pathways within the airways of a variety of species which are neither adrenergic nor cholinergic, the non-adrenergic, non-cholinergic (NANC) mechanisms. With respect to airway smooth muscle tone, NANC neural responses may induce either contraction (excitatory, e-NANC) or relaxation (inhibitory, i-NANC). Early investigations of NANC mechanisms in both human and other animal airways suggested a role for neuropeptides as the putative neurotransmitters. 2. Excitatory NANC (e-NANC) bronchoconstrictor responses are believed to be mediated by the release of sensory neuropeptides from a subpopulation of non-myelinated C-fibre primary afferent neurones in the airways. e-NANC nerves, which release tachykinins such as substance P (SP), neurokinin A (NKA) and the peptide calcitonin gene-related peptide (CGRP, produced as a result of alternative splicing of the calcitonin gene) are selectively degenerated by the nerve toxin capsaicin (an extract from hot peppers), with the subsequent abolition of the e-NANC responses. Tachykinin receptors have been detected by radio-ligand receptor binding studies and visualized by autoradiographic mapping, and exogenous addition of these peptides elicits a bronchoconstrictor response in both human and other animal airways. In addition to these effects on airway smooth muscle tone, tachykinins produce an increase in microvascular permeability (and associated oedema formation), mucus hypersecretion and cause an exaggerated cholinergic bronchoconstrictor response. Thus, tachykinins may play a role in the inflammatory process and contribute to the neurogenic inflammation as seen in asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2000

Reactive control of precision grip does not depend on fast transcortical reflex pathways in X-linked Kallmann subjects.

It has been shown that subjects maintain grasp stability by automatically regulating grip force in response to loads applied tangentially to a manipulandum held using a precision grip. Signals from cutaneous mechanoreceptors convey the information necessary for both the initiation and scaling of responses. The central neural pathways that support these grip reactions are unknown. However, the latency of the increase in force is similar to that of 'long-latency' transcortical reflexes recorded from muscles following muscle stretch or electrical stimulation of digital nerves. This study assessed the importance of fast transcortical pathways for reactive grip responses by examining these responses in subjects with X-linked Kallmann's syndrome (XKS). Subjects were selected whose corticospinal projection, as assessed by magnetic brain stimulation, is essentially ipsilateral, and in whom the long-latency reflex components following digital nerve stimulation are only found contralateral to the stimulated side. Despite this anomaly of the fast corticospinal pathway, these XKS subjects responded in the same way as control subjects; grip response latencies were similar and responses were appropriately scaled. However, the non-operating hand of these XKS subjects often mirrored the grip force changes of the operating hand. Reflex force mirroring was most marked during the first 50 ms and the force output was always less than 20 % of that of the operating hand. We conclude, firstly, that somatosensory driven precision grip responses that support grasp stability do not depend on fast conducting corticospinal pathways in these subjects and, secondly, that such responses do not use those 'long-latency' reflex pathways probed by cutaneomuscular reflexes elicited by electrical stimulation of digital nerves.

Adult↗

PubMed1996

Neural connections in and around the cavernous sinus in rat, with special reference to cerebrovascular innervation.

There is a confluence in and around the cavernous sinus of neural pathways innervating the intracranial structures. To determine the patterns of innervation, particularly of the cerebral arteries, we stained whole-mount preparations of the cavernous sinus and adjacent regions of the rat for acetylcholinesterase. The cavernous nerve plexus, with several small ganglia, mainly occupied the lateral wall of the sinus and extended laterally above the ophthalmic and maxillary divisions of the trigeminal nerve, in relation to the oculomotor and trochlear nerves. The cavernous plexus was connected to the pterygopalatine ganglion, the trigeminal ganglion, and the abducens nerve. The elongated pterygopalatine ganglion consisted of an orbital part, from which parasympathetic fibers ran to the cerebral arteries, and a cavernous part. Nerves from the lateral extension of the cavernous plexus ran rostrally into the orbit along the oculomotor, trochlear, and ophthalmic nerves, and caudally to the pineal gland along the trochlear nerve. Several branches also ran over the dura mater. Caudal to the cavernous sinus, we found two large nerves and a number of small nerves that ran between the nerves surrounding the internal carotid artery and the abducens nerve. These nerves may represent additional parasympathetic and/or sensory pathways to the cerebral arteries.

Animals↗

PubMed1999

Modulation of voiding and storage reflexes by activation of alpha1-adrenoceptors.

OBJECTIVE: This paper reviews recent studies in animals that examined the effect on lower urinary tract function of alpha1-adrenoceptor agonists and antagonists. METHODS: Bladder reflexes were studied in vivo on anesthetized rats and cats using cystometrographic and electrophysiologic techniques. Neurally-evoked bladder contractions and release of acetylcholine (ACh) were also studied in rat bladder strips in vitro. RESULTS: Administration of the alpha1-adrenoceptor agonist, phenylephrine (PE) to isolated strips of rat bladder enhanced neurally-evoked bladder contractions and increased basal tone. The former effects of PE were blocked by a selective alpha1A antagonist and the latter by an alpha1B antagonist. Activation of alpha1A receptors by PE enhanced ACh release evoked by electrical field stimulation in bladder strips. PE also enhanced transmission in cat bladder ganglia. PE or noradrenaline act on alpha- and beta-adrenoceptors on urothelial cells to release nitric oxide. It is concluded that facilitatory alpha1A-adrenoceptors are located prejunctionally in the bladder, whereas alpha1B adrenoceptors are located postjunctionally. In the central nervous system of the rat and cat facilitatory alpha1-adrenergic mechanisms can modulate the sympathetic, parasympathetic and somatic outflow to the urinary tract. In addition inhibitory alpha1 adrenoceptor mechanisms have been detected in the rat spinal cord. Activation of these receptors with PE raises the intravesical pressure threshold for inducing micturition and decreases voiding frequency. CONCLUSIONS: alpha1-adrenoceptors are located at various sites in the bladder and in the neural pathways controlling lower urinary tract function. At most sites these receptors mediate facilitatory responses that enhance smooth muscle activity or facilitate storage or voiding reflexes. However, alpha1-adrenoceptor inhibitory mechanisms in the rat spinal cord, can also reduce the frequency of voiding reflexes. This effect is possibly mediated by an inhibition in the afferent limb of the micturition reflex pathway.

Adrenergic alpha-Agonists↗

PubMed1975

Effects of estrogen on neuronal excitability in the hippocampal-septal-hypothalamic system.

The effects of electrical stimulation of the medial preoptic area (MPO) upon unit firing in the periventricular arcuate nucleus, and that of the dorsal hippocampus (DHPC) on medial septo-preoptic and arcuate (ARC) neurons, were investigated in Wistar and Sprague-Dawley female rats throughout the 4-day estrous cycle. Unit activity was recorded using stereotaxically-oriented tungsten microelectrodes under light urethane anesthesia. Repetitive stimulation of monophasic square waves varying only in current intensity was used. The following results were obtained: (a) An increase in activity of all ARC neurons recorded was induced by MPO stimulation on each day of the estrous cycle. (b) The minimum current (threshold) effective in increasing activity in the ARC neurons varied throughout the estrous cycle; the lowest threshold was observed in proestrus and the highest on the first day of diestrus. Also, the threshold current of MPO stimulation required in increase ARC activity was found to be elevated after ovariectomy and markedly reduced to the levels of the proestrous animal by estrogen treatment. (c) Stimulation of the DHPC (field CA3) increased activity in the medial septum but decreased activity in the MPO. Two pools of neurons, one increased and one decreased by DHPC stimulation, were observed in the ARC. (d) Variation in the threshold hippocampal stimulation during the estrous cycle was observed in the response of MPO and ARC neurons (the stimulation was effective only in proestrus and estrus); but not in that of medial septal neurons. (e) In addition, the spontaneous activity of septal; MPO; and ARC neurons was increased at proestrus or after estrogen injection. The present results suggest that plasma levels of estrogen play an essential role in the cyclic process of the regulation of ovulation by way of the selective facilitation of neuronal excitability in specific functional neural pathways. Furthermore, the results support the existence of a hippocampal inhibitory projection originating in field CA3 and terminating in the final common pathway, MPO to ARC.

Animals↗

PubMed1999

Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.

Animals↗