References

Richerson GB, Nattie EE, Deneris ES, Lauder JM (2003) Serotonergic neurons and development: implications for normal brain function and human disease. Neuroscience 2003 Abstracts 329. Society for Neuroscience, New Orleans, LA.

Summary: Symposium. Serotonergic neurons have widely divergent projections to virtually all of the CNS, and are involved in a variety of brain functions. This symposium will focus on how dysfunction of 5-HT neurons during development can influence brain function throughout life. G Richerson will discuss pH chemosensitivity of 5-HT neurons, how this changes during development, and the emerging hypothesis that these neurons induce arousal, a feeling of suffocation and hyperventilation in response to increased CO2. E Nattie has used focal manipulations of the raphe in vivo, including cell specific killing with an antibody to the serotonin transporter conjugated to the toxin saporin, to show that dysfunction of 5-HT neurons may lead to a defect in physiologic regulatory processes that are important during development. E Deneris will discuss mutant mice lacking the Pet-1 ETS gene, in which the majority of CNS 5-HT neurons are missing. 25-30% of Pet-1 nulls die during the first postnatal week, which may result from abnormal respiration. Surviving adults display anxiety-like and aggressive behavior. J Lauder will discuss 5-HT as a differentiation signal in prenatal brain development and as a morphogen in craniofacial development. Effects of prenatal exposure to serotonergic drugs or neurotoxins on postnatal outcome will be described. The speakers will introduce new hypotheses about how dysregulation of 5-HT neurons and 5-HT receptors during development may lead to a variety of brain disorders such as SIDS, migraine, autism, panic attacks and anxiety.

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Cooper-Kuhn CM, Winkler J, Kuhn H (2003) Altered neurogenesis after cholinergic forebrain lesion in the adult rat. Neuroscience 2003 Abstracts 348.9. Society for Neuroscience, New Orleans, LA.

Summary: Adult hippocampal neurogenesis has been shown to be functionally connected to learning and memory and at the same time to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic forebrain system is one of the key transmitter systems for learning and memory. Within the hippocampus and olfactory bulb, two regions of adult neurogenesis, cholinergic innervation is quite extensive. This experiment aims at defining the role of cholinergic input during adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion the cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the dentate gyrus and the olfactory bulb. Five weeks after lesion the rate of neurogenesis declined significantly in the dentate gyrus and olfactory bulb granule cell layers, whereas the generation of neurons in the periglomerular region of the olfactory bulb was unaffected. The number of apoptotic cells increased specifically in the progenitor region of the dentate gyrus as well as in the periglomerular layer of the olfactory bulb. Therefore, one of the possible mechanisms by which acetylcholine could promote neurogenesis is by increasing the survival of progenitor and immature neurons. Neurotransmitters can alter the microenvironment of neural progenitor cells, whether directly or indirectly, and these changes lead to significant alterations in neurogenesis. In principle, the data suggest that acetylcholine is stimulatory to adult hippocampal neurogenesis, since neurotoxin lesions specific to this neurotransmitter system lead to a reduced number of new neurons.

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Co C, Yin X, Johnson WE, Martin TJ, Smith JE (2003) The effects of IgG-192-saporin lesions of limbic forebrain on rat cocaine self-administration. Neuroscience 2003 Abstracts 422.3. Society for Neuroscience, New Orleans, LA.

Summary: The involvement of cholinergic neurons in cocaine self-administration has been recently demonstrated. This study was undertaken to further assess the role of cholinergic innervations of/ or interneurons in limbic brain regions previously shown to receive enhanced dopamine input during cocaine self-administration. Rats were trained to self-administer cocaine on an FR2 schedule using a within session dose intake procedure (3½ hour session with 1 hour access each to 0.17, 0.33 and 0.67 mg/infusion). The doses were then decreased systematically to threshold levels where only the highest dose was self-administered during the session. The cholinergic neurotoxin IgG-192-saporin (0.25 µg in 1 µl) or vehicle was then bilaterally administered into the posterior nucleus accumbens (NAcc) – ventral pallidum (VP). The saporin lesion resulted in a shift to the left in the dose intake relationship for cocaine self-administration with all three doses maintaining responding. The sham-vehicle treated rats continued to only sample the higher dose. Real time RT-PCR was used to assess the magnitude and extent of the lesion. Gene expression for p75 (the target for 192 IgG) and choline acetyltransferase (ChAT) were assessed in the NAcc, VP, caudate nucleus (CP) and diagonal band (DB) of these rats. Significant reductions in p75 and ChAT gene expression were seen in the DB and VP while only small decreases were seen in the NAcc and CP of the saporin treated rats. These data suggest that the overall influence of cholinergic neurons in the DB and VP are inhibitory to the processes underlying cocaine self-administration.

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Parikh T, Lee S, Walker BR (2003) Inhibition within the nucleus tractus solitarius (NTS) ameliorates social deficits due to specific acetylcholine (ACh) or Purkinje cell lesions. Neuroscience 2003 Abstracts 423.18. Society for Neuroscience, New Orleans, LA.

Summary: Previously, we demonstrated that enhancement of GABA transmission, or blockade of ionotropic glutamate within rat brainstem structures, which mediate limbic-motor seizure control, attenuated behavioral deficits, which were similar to those seen in human patients with autism, due to developmental cerebellum lesions. Evidence suggests that within autism spectrum disorders, there is a decrease in cholinergic neurons in the forebrain and/or a loss of purkinje cells in the cerebellum which might account for these behavioral deficits. Therefore, in the present study, we tested the hypothesis that specific lesions to the rat ACh system or reduction of purkinje cells in the rat cerebellum would lead to specific alterations of social behavior. Furthermore, alterations in GABA and glutamate transmission within the NTS would correct these social deficits. We examined the effect of ACh or purkinje cell lesions on social behavior in rats by recording social interactions before and after bilateral saporin injections (192-IgG or OX-7; 2 µg/side). As compared to preinjection behavior, saporin injections decreased social interaction of adult rats. Bilateral microinjections of the GABA agonist muscimol (256 pmol) into the mNTS at least 10 minutes prior to behavioral testing returned the amount of social investigation of the lesioned animals to pre-saporin levels. These findings suggest that specific neuronal populations are responsible for mediating social behavior in rats, and that there is a functional connection between those systems and the brainstem structures utilized for seizure control.

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Ukairo OT, Arshad S, Gibbs RB, Johnson DA (2003) Selective cholinergic lesion of the medial septum impairs retention but not acquisition of a passive avoidance memory task. Neuroscience 2003 Abstracts 425.16. Society for Neuroscience, New Orleans, LA.

Summary: Infusion of 192 IgG-saporin (SAP) into the medial septum (MS) selectively destroys cholinergic neurons projecting to the hippocampus. This study examined the effect of such lesions on acquisition and retention using a passive avoidance paradigm. Male Sprague-Dawley rats received either SAP (.22 μg in 1 μl) or vehicle directly into the MS. Passive avoidance training began two weeks later. Training consisted of placing an animal into the lighted chamber of the apparatus and then delivering footshock (.75 mA, 1 sec.) when the animal moved into the adjacent darkened chamber. Training was repeated until animals avoided the dark chamber for 2 consecutive trials of 5 min. duration. Retention (latency to enter the dark chamber) was tested 1 week later. Results showed no effect of SAP lesion on the number of trials necessary to acquire avoidance behavior. In contrast, SAP-lesioned animals showed a significant impairment in retention, as evidenced by a 72% decrease in crossover latency one week following training. These results suggest that selective destruction of cholinergic septo-hippocampal projections impairs retention, but not acquisition, of passive avoidance behavior to aversive stimuli.

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Fitz NF, Gibbs RB, Johnson DA (2003) Arousal enhances delayed match to position T-maze performance independent of septo-hippocampal cholinergic projections. Neuroscience 2003 Abstracts 425.17. Society for Neuroscience, New Orleans, LA.

Summary: Infusion of the selective cholinergic immunotoxin, 192 IgG-saporin (SAP) into the medial septum (MS) of rats selectively lesions cholinergic neurons projecting to the hippocampus and impairs acquisition of a delayed matching to position (DMP) T-maze task. The intent of the present study was to determine if enhanced performance associated with arousal is dependent on septo-hippocampal cholinergic projections. Male Sprague-Dawley rats received MS infusions of SAP 0.22 µg in 1µl or vehicle. Fourteen days later, animals were trained on the DMP spatial memory task. SAP and control animals were randomized into an “arousal” group that was injected with saline (IP; 1 ml/Kg) 30 min before testing each day or a “non-arousal” group that was not. SAP lesions significantly impaired acquisition of the DMP task in both the arousal and non-arousal groups. Conversely, arousal significantly enhanced acquisition in both control and SAP lesioned rats. There was no significant interaction between the effects of cholinergic lesions and arousal. These results suggest that septo-hippocampal cholinergic projections are not engaged in enhanced spatial learning mediated by arousal.

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McGaughy JA, Jindal M, Eichenbaum HB, Hasselmo ME (2003) Cholinergic deafferentation of the entorhinal cortex in rats impairs encoding of novel but not familiar stimuli in a delayed non-match to sample task (DNMS). Neuroscience 2003 Abstracts 425.4. Society for Neuroscience, New Orleans, LA.

Summary: Muscarinic cholinergic receptor activation in entorhinal cortex (EC) activates intrinsic depolarizing membrane currents which cause self-sustained spiking activity in single neurons (Klink and Alonso, J. Neurophys. 77, 1997). This effect may underlie delay activity and match-dependent activity changes in delayed match to sample tasks (Fransen et al., J. Neurosci. 22, 2002) and could allow accurate maintenance of novel information without dependence on synaptic modification associated with previous exposure (familiarization). Consistent with this, research in human subjects suggest that the medial temporal lobes are specifically activated during working memory for novel but not familiar stimuli (Stern, et al. Hippocampus v. 11, 2001), and cholinergic deafferentation of the rhinal cortex in non-human primates has been shown to impair memory for trial-unique (novel) stimuli (Turchi et al., SFN abstracts v. 28). The current study tests the hypothesis that cholinergic deafferentation of the EC produces impairments in working memory for novel but not familiar stimuli. Prior to surgery rats were trained in an odor DNMS task with a brief delay. After reaching asymptotic performance, rats were infused with either 192-IgG-saporin (SAP) or its vehicle into the EC (0.01 µg/µl; 1.0 µl/injection; 6 infusions/hemisphere). Rats were not impaired at any delay when tested with familiar odors but showed significant, persistent impairments when tested with novel odors. An increase in task difficulty alone was insufficient to explain these effects. These data support the hypothesis that cholinergic afferents to the EC activate cellular mechanisms of sustained spiking activity necessary for maintenance of novel but not familiar stimuli in a working memory task. Support Contributed By: NIH MH61492, MH60013, DA16454.

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Butt AE, Corley S, Cabrera S, Chavez C, Kitto M, Ochetti D, Renovato A, Salley T, Sarpong A (2003) 192 IgG-saporin lesions of the nucleus basalis magnocellularis in rats fail to disrupt acquisition or retention of differential reinforcement of low rate responding. Neuroscience 2003 Abstracts 425.5. Society for Neuroscience, New Orleans, LA.

Summary: The frontal cortex has been implicated in supporting timing behavior in tests of differential reinforcement of low rate responding (DRL) in rats. DRL performance is similarly influenced by anticholinergic drugs; scopolamine interferes with DRL performance by increasing the number of nonreinforced responses and thus decreasing DRL efficiency. Because the frontal cortex receives significant cholinergic input from the nucleus basalis magnocellularis (NBM) in rats, we hypothesized that NBM lesions would disrupt DRL performance in the current experiment. Male Long-Evans rats were placed first in a DRL 10 s schedule of reinforcement before advancing to a DRL 20 s schedule. Rats received 50 trials per day for 20 consecutive days on both DRL schedules. When rats reached stable performance, they received either bilateral 192 IgG-saporin lesions of the NBM or sham lesions. Upon recovery, rats were reintroduced to the DRL 20 s task for 10 days of post-operative testing. Finally, rats were tested using a novel delay interval in a DRL 30 s task. Testing continued for 10 additional days. Results showed that the NBM lesion group showed no significant change in either the total number of responses or in DRL efficiency (reinforced responses / total responses) between pre- and post-operative DRL 20 s testing. Subsequent acquisition in the DRL 30 s task was similarly not disrupted by NBM lesions. The effectiveness of the lesions was verified by acetylcholinesterase (AChE) staining, which showed pronounced depletion of cortical AChE with normal AChE-positive staining in the hippocampus and medial septal area. These data suggest that the NBM is not critically involved in either the acquisition or retention of DRL performance.

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Wiley RG, Miller SA, Kline IV RH (2003) Selective destruction of MOR expressing dorsal horn neurons using intrathecal dermorphin-saporin. Neuroscience 2003 Abstracts 174.15. Society for Neuroscience, New Orleans, LA.

Summary: Evidence suggests that the mu opiate receptor (MOR) is key to the analgesic action of morphine. In the present study, we sought to determine if a disulfide conjugate of the mu opioid peptide, dermorphin, to the ribosome-inactivating protein, saporin, (derm-sap) would destroy neurons expressing MOR in the substantia gelatinosa (SG) of the spinal cord. Derm-sap was injected into the lumbar subarachnoid space of anesthetized adult, male Sprague-Dawley rats using a catheter inserted through the atlanto-occipital membrane and passed 8 cm caudally. The catheter was removed 15 minutes after toxin injection. Rats were sacrificed after 2 weeks, and 40 um transverse frozen sections of the L4 spinal segment were processed for immunohistochemical demonstration of MOR, NeuN, calbindin D28k, parvalbumin, NK-1R and for Nissl staining. In control rats, beta-funaltrexamine was injected just before derm-sap or derm-sap was pre-treated to reduce the disulfide bond which dissociates the toxin and neuropeptide. MOR staining in the SG was evaluated using quantitative densitometry. Initial experiments revealed a dose-related decrease in MOR staining in the dorsal horn without effect on dorsal root ganglia at doses up to 1000 ng. The maximally tolerated dose of derm-sap (500 ng) selectively decreased MOR staining by 54% as did multilevel lumbar dorsal rhizotomy. Combining 500 ng of derm-sap and multilevel rhizotomy produced 92% loss of MOR staining in the SG. Based on analysis of non-co-localized markers and control experiments, we interpret the results to indicate that intrathecal derm-sap selectively destroys MOR-expressing neurons in the SG without toxicity to primary afferents. This lesion will be useful in analysis of opioid mechanisms in the dorsal horn.

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Kline IV RH, Wiley RG (2003) SSP-saporin decreases formalin induced c-Fos expression throughout the dorsal horn. Neuroscience 2003 Abstracts 174.7. Society for Neuroscience, New Orleans, LA.

Summary: Substance P (SP) antagonists and SP-saporin have been shown to decrease phase II of the formalin test suggesting an important role for SP in this model of persistent pain. SP antagonists also decrease formalin induced c-fos expression in dorsal horn neurons. A congener of SP-sap that is more stable and has higher affinity for NK-1R, SSP-sap (Sar9Met(02)11-substance P-saporin) has been studied by injection into the striatum and hippocampus where it was more potent and specific than SP-sap. In the present study, this selective and more potent toxin was used to determine the effects of destroying dorsal horn NK-1R on behavior and c-fos induction after intraplantar formalin. Twelve Sprague Dawley male rats were injected intrathecally with 100ng SSP-sap or PBS. After 2 weeks survival, rats underwent hindpaw formalin injections and behavioral scoring, and then were sacrificed after 3 hours and the lumbar spinal cords processed for immunohistochemical demonstration of NK-1R and c-fos. There were significant correlations between the loss of superficial laminae NK-1R neurons, decreased formalin behavior and dorsal horn c-fos expression. Therefore lumbar i.t. SSP-sap 1) decreased NK-1R cells in laminae I but not in the deeper laminae 2) decreased phase II formalin behavior 3) decreased c-fos in both the superficial and deep laminae. Since c-fos expression in the deeper laminae was decreased and NK-1R was spared in these laminae, we conclude that a lesion affecting only laminae I NK-1R lesion alters activation of neurons throughout the dorsal horn suggesting a key role for the missing neurons in the transfer of nociceptive inputs to deeper laminae.

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