Repeated intracerebral teneurin C-terminal associated peptide (TCAP)-1 injections produce enduring changes in behavioral responses to corticotropin-releasing factor (CRF) in rat models of anxiety

The teneurin C-terminal associated peptides (TCAP) are a recently discovered family of peptides encoded by a bioactive neuropeptide-like gene sequence found at the carboxy terminus of the teneurin transmembrane proteins. TCAP is structurally related to the corticotropin-releasing factor (CRF) family of peptides. Synthetic TCAP-3 and TCAP-1 are active in vitro in stimulating cAMP and proliferation in neuronal lines. TCAP-1 mRNA is expressed in limbic brain regions and modulates acoustic startle behavior in rats when injected into the basolateral amygdala. In the current study, TCAP-1 was administered into the cerebral ventricles once per day for 5 days to rats. At 1-3 weeks after the last TCAP-1 treatment, the rats were tested in the elevated plus maze, open field test, or the acoustic startle test, with or without an acute CRF injection 30 min prior to the test. The results show a difference in behavioral response between TCAP-treated and saline-treated rats, but only when an acute CRF challenge is delivered prior to testing. In the plus maze and open field tests, acute CRF effects were enhanced by prior TCAP-1 treatment, whereas in the acoustic startle test, the acute CRF effects were diminished by prior TCAP-1 administration.     

Excitatory amino acid antagonists depress acoustic startle after infusion into the ventral nucleus of the lateral lemniscus or paralemniscal zone

Rats were implanted with bilateral cannulas in an area just medial to the ventral nucleus of the lateral lemniscus, an obligatory relay along the acoustic startle pathway. Bilateral infusions of excitatory amino acid transmitter antagonists into this region (10, 25 or 50 nmol per side) produced a rapid, dose-dependent depression of acoustic startle. gamma-D-Glutamylglycine, gamma-D-glutamylaminomethyl sulfonate and 2-amino-5-phosphonovalerate were equally effective in depressing the startle response over this dose range. These results indicate that excitatory amino acid transmitters play an important role in the expression of acoustic startle at this part of the startle pathway.     

Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein

A rodent cortex-enriched mRNA, RC3, was identified by screening a rat brain cDNA library with a cortex-minus-cerebellum subtracted cDNA probe. Conceptual translation of RC3 cDNA sequences indicates that the rat and mouse mRNAs encode identical, novel 78 amino acid proteins. The RC3 protein amino terminus contains a cysteine-rich domain similar to those found in snake venom neurotoxins; the carboxyl terminus contains a collagen-like motif that may function in the assembly of RC3 subunits into a multimeric protein. Western blot experiments with an antiserum to a synthetic peptide corresponding to 27 residues of the 78 residue sequence identify an immunoreactive polypeptide with 18 kDa gel mobility that is likely to correspond to the RC3 protein. Northern blot analysis and in situ hybridization experiments show that RC3 mRNA is highly enriched in rat brain, with restricted expression in neuronal subsets primarily in the cortex, striatum, and hippocampus as well as certain nuclei within the thalamus, hypothalamus, the olfactory bulb.     

Lipopolysaccharide produces dose-dependent reductions of the acoustic startle response without impairing prepulse inhibition in male rats

This study examined the dose-dependent effects of Lipopolysaccharide (LPS) on the acoustic startle response and prepulse inhibition (PPI) in male Long-Evans rats. LPS is known to stimulate the innate immune system and result in behavior modifications referred to as "sickness behaviors". The purpose of this study was to assess the ability of LPS to modulate sensorimotor reflexes (Startle-Only trials) and/or sensory processing (PPI trials). Rats were injected intraperitoneally with LPS (50, 100 or 200 microg/kg LPS, n=9/group) or saline vehicle (n=14) on 2 test days 72 h apart. Subjects were placed in a familiar startle box apparatus where startle response magnitudes were recorded following 115 dB Startle-Only trials and PPI trials (with prepulses at +3, +6 and +12 dB above background noise). Analysis of Startle-Only trials indicated a significant dose-dependent effect of LPS on Test Day 1. The 200 microg/kg LPS group exhibited significantly reduced startle response magnitude relative to all other treatments. On the PPI trials no LPS groups displayed significantly different performance from vehicle controls. Also, DayxDrug interactions for both Startle-Only and PPI trial types indicated behavioral tolerance to LPS. LPS reduced the acoustic startle response in a dose-dependent manner on Test Day 1. From the PPI data, it is evident that all treatment groups elicited near-normal inhibition levels indicating adequate sensory function. In combination, the results suggest that the range of sickness behaviors following LPS-administration to adult rats includes decreased non-voluntary motor activity as reflected by reduced startle magnitude.     

Acquisition of acoustic startle shows a dose-response to serum free T4

Methimazole, administered to rats in drinking water (0.1 and 0.05%) from embryonic day 17 to postnatal day 10, caused a dose-dependent decrease in serum Free T4 and an accompanying dose dependent delay in acquisition of acoustic startle reflex. In conjunction with other studies showing the specific dependence of acoustic startle development on thyroid hormone, this study suggests that the acoustic startle system may be a useful model for determining thyroid hormone requirements for normal neurologic development.     

Moxonidine, a selective imidazoline-1 receptor agonist, suppresses the effects of ethanol withdrawal on the acoustic startle response in rats.

BACKGROUND: There is a need for improved treatments for ethanol withdrawal in humans. Previously, ethanol withdrawal has been shown to enhance the acoustic startle response in rats. Because many ethanol withdrawal symptoms are caused by autonomic hyperactivity, we examined the effects of two antihypertensives, the imidazoline(I)(1) agonist moxonidine and the alpha(2)-adrenergic partial agonist clonidine, on the ethanol-withdrawal-enhanced acoustic startle response in rats. d-amphetamine-enhanced startle served as a positive control. METHODS: Male, Long-Evans rats were made ethanol-dependent through unlimited access to liquid diet containing 6.7% v/v ethanol for 10 days. The concentration of ethanol was reduced to 3.3% v/v on the 11th day. On the 12th day, the rats received control diet. The acoustic startle response was tested 24 hours following the withdrawal of ethanol. Control rats were maintained on control liquid diet throughout the experiment. RESULTS: As has been shown previously, withdrawal from the chronic ingestion of ethanol significantly enhanced the acoustic startle response. Pretreatment with moxonidine (0.01, 0.1, and 1.0 mg/kg, subcutaneously), but not clonidine (0.3, 1.0, and 3.0 mg/kg, subcutaneously), significantly attenuated the ethanol withdrawal-induced elevation of the acoustic startle response. Moxonidine did not suppress the elevation in the startle response caused by d-amphetamine. CONCLUSIONS: These results indicate that I(1) receptors can play an important role in ethanol withdrawal and that moxonidine may be useful for the treatment of ethanol withdrawal in humans.     

Attenuation of auditory startle and prepulse inhibition by unexpected changes in ambient illumination through dopaminergic mechanisms

We investigated the role of dopaminergic mechanisms in the attenuation of the acoustic startle response and prepulse inhibition (PPI) in rats by the introduction of unexpected changes in environment illumination. Experiment 1 showed that Dark-to-Light transitions robustly reduce startle responses and PPI. Experiment 2 showed that this phenomenon habituates across repeated testing sessions and reappears after an interval without testing. Experiment 3 demonstrated that haloperidol blocks the startle and PPI-reducing effect of the Dark-to-Light transition. We show how a computational model of acoustic startle response and prepulse inhibition can be extended to incorporate the empirical effects demonstrated in this study. We conclude that sensory gating as measured by prepulse inhibition is markedly attenuated in situations where novel stimuli are introduced during a test session and that dopaminergic systems may be involved in the dynamic changes evoked by the onset of illumination.     

NMDA and non-NMDA antagonists infused into the nucleus reticularis pontis caudalis depress the acoustic startle reflex

The neural pathway that mediates the acoustic startle reflex has been proposed; however, the pharmacology underlying this reflex is less well known. The present study examined the role of excitatory amino acid receptors at the level of the nucleus reticularis pontis caudalis, a brainstem nucleus obligatory for the whole body startle reflex and implicated as the locus where extrinsic systems such as the amygdala may act to modulate acoustic startle. Twenty-nine rats, chronically implanted with bilateral cannulae aimed at the nucleus reticularis pontis caudalis, were tested to assess the effects of γ-d-glutamylglycine (DGG),dl-2-amino-5-phosphonopentanoic acid (AP5), and 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX) on the amplitude of the acoustic startle reflex. Local infusion of each of the 3 compounds significantly reduced startle amplitude by as much as 70–80%. AP5 and CNQX attenuated startle over a dose range which indicated that the reticularis pontis caudalis may be much more sensitive to these compounds than other nuclei along the primary startle pathway. These results suggest that, at the level of the nucleus reticularis pontis caudalis, an excitatory amino acid neurotransmitter may mediate acoustic startle, and that both NMDA and non-NMDA receptor subtypes may be important for the expression of the acoustic startle reflex.     

Acoustic startle amplitude predicts vulnerability to develop post-traumatic stress hyper-responsivity and associated plasma corticosterone changes in rats

Following exposure to trauma, a vulnerable sub-population of individuals develops post-traumatic stress disorder (PTSD) with characteristic persistent autonomic hyper-responsivity, associated increased startle response, and commonly altered hypothalamo-pituitary-adrenal regulation. A goal of this investigation was to identify a predictive marker for this vulnerability. Previous investigators have developed a model for PTSD in which male mice were exposed to a single brief episode of inescapable footshock followed by 1-min contextual reminders of this trauma at weekly intervals for 6 weeks. Exposure to these reminders induced a progressive and persistent increase in the amplitude of acoustic startle consistent with the persistently increased acoustic startle of individuals exhibiting PTSD. We adapted this model to adult male Wistar rats, with added characterization of initial (pre-trauma) startle response. After one episode of inescapable footshock (10 s, 2 mA) or control treatment followed by six weekly 1-min contextual reminders, acoustic startle was re-tested. Data were analyzed after dividing rats within each treatment into LOW vs MID vs HIGH (33% in each group) pre-treatment startle responders. Rats which exhibited pre-treatment LOW- and MID-range acoustic startle responses did not develop increased acoustic startle responses following subsequent traumatic stress+reminders ([TS+R]) treatment. However, rats which exhibited HIGH pre-treatment startle responses exhibited further significant (p<0.01) [TS+R]-induced persistent enhancement of this already elevated startle response. Furthermore, rats exhibiting HIGH pre-treatment startle responses were also the only subgroup which exhibited increased basal plasma corticosterone levels following [TS+R] treatment. These results suggest that initial pre-stress acoustic startle response can identify subgroups of rats which are predisposed to, or resistant to, developing a PTSD-like syndrome following subsequent trauma.     

Involvement of cyclic AMP at the level of the nucleus reticularis pontis caudalis in the acoustic startle response

Rats were implanted with cannulas in the nucleus reticularis pontis caudalis (PnC), an obligatory part of the neural pathway that mediates the acoustic startle reflex. Following at least 1 week of recovery, rats were tested for acoustic startle amplitude before or after infusion of compounds known to alter the second messenger, adenosine cyclic 3′, 5′-monophosphate (cAMP). Local infusion into the PnC of the cAMP analog, 8-bromo cAMP (0.125–1.0 μg), increased the amplitude of the acoustic startle response in a dose-dependent manner. In addition, local infusion of a phosphodiesterase inhibitor, rolipram (10 μg) or the water soluble adenylate cyclase activator, forskolin-DHA (2.5 μg), produced a significant enhancement of startle amplitude. These effects probably resulted from intracellular actions because cAMP itself, which does not readily penetrate lipid membranes, had no effect. Moreover, the effects seemed somewhat specific because the precursor of cAMP, ATP or 8-bromo cGMP, also failed to alter startle at doses where 8 bromo-cAMP did. The fact that a phosphodiesterase inhibitor elevated startle suggests that cAMP serves to tonically elevate startle at this level of the pathway. Hence, treatments that either increase (fear, sensitization) or decrease (habituation, pre-pulse inhibition) startle at the level of the PnC may do so via release of neurotransmitters either positively or negatively coupled to cAMP, which in turn may alter either sound evoked transmitter release, excitability of PnC neurons or both.     

Footshock-induced sensitization of the acoustic startle response in two strains of mice

It has been shown before that unconditioned footshocks can augment the acoustic startle response in rats. In the present study, male mice of two strains, C57Bl/6N and BALB/c, were compared with regard to footshock-induced sensitization of the acoustic startle response. Presentation of footshocks did not affect the acoustic startle response in C57Bl/6N mice, while in contrast, footshock-induced sensitization was apparent in the BALB/c strain. Shocked C57Bl/6N mice, but not BALB/c mice, displayed robust conditioning to the startle context when re-tested the next day. These findings indicate that mice may exhibit footshock-induced sensitization of the acoustic startle response, but that the effects of footshocks on the acoustic startle are strain- and time-dependent.     

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) ord,l-2-amino-5-phosphonovaleric acid (AP-5)

The present study investigated the individual contributions of spinal cord N-methyl-d-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist,d,l-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterize the effects of these two compounds on the early ( 8ms) or late ( 15ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.     

Differential blockade of early and late components of acoustic startle following intrathecal infusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-2-amino-5-phosphonovaleric acid (AP-5)

The present study investigated the individual contributions of spinal cord N-methyl-D-aspartate (NMDA) and non-NMDA receptors to the acoustic startle reflex in rats. The first experiment measured whole body acoustic startle before and after intrathecal infusion of various doses of either the NMDA receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (AP-5), or the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Both compounds depressed startle in a dose-dependent fashion with similar potencies. A second experiment measured startle electromyographically (EMG) in the quadriceps femoris muscle complex in the hindlimbs during auditory stimulation to characterized the effects of these two compounds on the early (approximately 8 ms) or late (approximately 15 ms) EMG components of the startle response. CNQX preferentially blocked the early EMG component of startle, whereas AP-5 preferentially blocked the late component. These results suggest that the acoustic startle reflex involves an early EMG component mediated by spinal non-NMDA receptors, and a late EMG component mediated by spinal NMDA receptors.     

Neonatal herpes simplex virus type 1 brain infection affects the development of sensorimotor gating in rats

The effect of neonatal brain infection of herpes simplex virus type 1 (HSV-1) on the development of sensorimotor function in the rat was investigated using an acoustic startle paradigm. Intracerebral inoculation of HSV-1 at day 2 after birth, but not at day 4, caused a significant delay in the development of prepulse inhibition of acoustic startle. A decrease in prepulse inhibition was shown at 37, 46 and 58 days of age in these rats compared to control rats. No evidence was obtained for other behavioural dysfunctions such as differences in sensorimotor reactivity, sensorimotor response habituation, spontaneous locomotor activity, rearing activity or stereotyped behaviour. Prepulse inhibition of acoustic startle is an accepted model of sensorimotor gating in the CNS, a function which has been shown diminished in schizophrenic persons. The present results suggest that early viral infections during a neurone-susceptible period may contribute to the development of this deficit.     

The effects of intra-accumbens neurotensin on sensorimotor gating

Neurotensin is a neuropeptide which coexists with mesolimbic dopamine. Previous studies have shown that centrally administered neurotensin can modulate the activity of mesolimbic dopamine with a profile similar to neuroleptics. For example, infusions of neurotensin into the nucleus accumbens inhibit amphetamine-induced hyperlocomotion. Prepulse inhibition (PPI) occurs when a weak prestimulus (‘prepulse') inhibits the amplitude of the startle response to an intense stimulus (‘pulse'). PPI is an operational measure of sensorimotor gating which is strongly regulated by mesolimbic dopamine. This study examined the effects of various doses of neurotensin infused into the nucleus accumbens of rats on the prepulse inhibition (PPI) of their acoustic startle reflex. Neurotensin (0.25–5.0 μg) was infused into the nucleus accumbens of rats. Animals then received subcutaneous injections of amphetamine (2 mg/kg) or saline and were placed in startle chambers where measures of startle amplitude and PPI were obtained. Neurotensin increased baseline PPI and blocked amphetamine-induced disruption of PPI in a dose-dependent fashion. The lowest dose of neurotensin tested (0.25 μg) significantly increased baseline PPI and both 0.25 and 1.0 μg neurotensin blocked amphetamine-induced decreases in PPI. The 5.0 μg dose of neurotensin had no significant effect on prepulse inhibition. Neurotensin had no effect on the amplitude of the acoustic startle reflex in amphetamine- or saline-treated rats. The results suggest that intra-accumbens neurotensin has a significant, dose-dependent effect on sensorimotor gating in which lower doses (0.25–1.0 μg) exhibit a neuroleptic-like action.     

A novel role of the corticotrophin‐releasing hormone regulating peptide,teneurin C‐terminal associated peptide 1, on glucose uptake into the brain

Teneurin C‐terminal associated peptide (TCAP) is an ancient paracrine signalling agent that evolved via lateral gene transfer from prokaryotes into an early metazoan ancestor. Although it bears structural similarity to corticotrophin‐releasing hormone (CRH), it inhibits the in vivo actions of CRH. The TCAPs are highly expressed in neurones, where they induce rapid cytoskeletal rearrangement and are neuroprotective. Because these processes are highly energy‐dependent, this suggests that TCAP has the potential to regulate glucose uptake because glucose is the primary energy substrate in brain, and neurones require a steady supply to meet the high metabolic demands of neuronal communication. Therefore, the objective of the present study was to assess the effect of TCAP‐mediated glucose uptake in the brain and in neuronal cell models. TCAP‐mediated ¹⁸F‐deoxyglucose (FDG) uptake into brain tissue was assessed in male wild‐type Wistar rats by functional positron emission tomography. TCAP‐1 increased FDG uptake by over 40% into cortical regions of the brain, demonstrating that TCAP‐1 can significantly enhance glucose supply. Importantly, a single nanomolar injection of TCAP‐1 increased brain glucose after 3 days and decreased blood glucose after 1 week. This is corroborated by a decreased serum concentration of insulin and an increased serum concentration of glucagon. In immortalised hypothalamic neurones, TCAP‐1 increased ATP production and enhanced glucose uptake by increasing glucose transporter recruitment to the plasma membrane likely via AKT and mitogen‐activated protein kinase/ERK phosphorylation events. Taken together, these data demonstrate that TCAP‐1 increases glucose metabolism in neurones, and may represent a peptide signalling agent that regulated glucose uptake before insulin and related peptides.     

Induction of severe experimental autoimmune neuritis with a synthetic peptide corresponding to the 53-78 amino acid sequence of the myelin P2 protein.

We generated a synthetic peptide (SP-26), corresponding to the amino acid residues 53-78 of bovine P2 protein, which induced severe clinical and pathological characteristics of experimental autoimmune neuritis (EAN) in Lewis rats. Lymph node cell populations from SP-26-immunized rats elicited a proliferative response to the peptide and to the P2 protein. After 16 cycles of antigen stimulation with the peptide, the SP-26 T cell line shows a decreased response to P2, but not to SP-26. Fluorescence-activated cell sorter (FACS) analysis of a SP-26 T cell line indicated the majority of cells to be of CD4+ CD8-. This report demonstrates that the synthetic peptide SP-26 can induce severe EAN in Lewis rats in a dose-dependent manner. Furthermore, specific T cell lines reactive to SP-26 can be generated from the lymph nodes of SP-26-immunized rats.     

Neonatal Borna disease virus infection (BDV)-induced damage to the cerebellum is associated with sensorimotor deficits in developing Lewis rats

Neonatal Borna disease virus (BDV) infection of the brain produces developmental damage to the cerebellum in Lewis rats, with minimal classical inflammatory responses. In the present study, we assessed the consequences of this damage by measuring motor coordination and postural skills in developing (postnatal days 4 to 30) Lewis rats that were neonatally infected with BDV. Neonatal BDV infection-induced motor impairments were selective and correlated with the time course of BDV damage to cerebellar development. BDV-induced motor deficits were not seen until the end of postnatal week 2. By postnatal week 3, BDV-infected rats had deficits in negative geotropism, fore- and hind limb placing and grasping. BDV-infected rats also exhibited deficits in the ability to hold on to a bar and to cross a suspended bar. Neonatal BDV infection induced impairments in the acoustic startle response. Compared to controls, neonatally BDV-infected rats exhibited attenuated habituation of the acoustic startle at postnatal day (PND) 23 and decreased startle responsiveness at PND 30. Prepulse inhibition of the acoustic startle remained unaltered in BDV-infected rats. The data demonstrate that neonatal BDV brain infection of rats can be a valuable animal model system for studying the relationship between abnormal brain development and resultant behavioral deficits. Further studies of this model may elucidate specific pathogenic mechanisms that that may have implications in the study of neurodevelopmental human disorders.     

Stress-induced enhancement of auditory startle: an animal model of posttraumatic stress disorder

An innovative animal model of posttraumatic stress disorder (PTSD) is proposed in which nonhabituation of the acoustic startle response is developed in rats subsequent to tailshock exposure. Subjects (n = 31) received 30 minutes of intermittent tail shock on 2 days followed by exposure to the tailshock apparatus on the third day. Compared to baseline startle reactions, 9 of 31 tailshock-exposed rats developed nonhabituation of startle response reactions during the subsequent 3 weeks of testing. No control rats developed nonhabituation of startle reactions over a similar time period. These data suggest that this system models useful aspects of clinical PTSD emphasizing nonhabituation of startle reactions as a dependent variable. The method consistently identifies a subgroup of rats that develop persistent nonhabituation of startle in response to a tailshock-stress paradigm.