Stanley A. Lorens

Open field activity and avoidance behavior following serotonin depletion: A comparison of the effects of parachlorophenylalanine and electrolytic midbrain raphe lesions

Three experiments were performed in order to compare the behavioral effects of electrolytic destruction of the dorsal and median mesencephalic raphe nuclei (MR lesion) and parachlorophenylalanine (pCPA; 300 mg/kg, IP) administration. Forebrain 5-hydroxytryptamine (5-HT) was measured in all animals following completion of behavioral testing. In the first experiment open field behavior (one 50 min session) and two-way (shuttle) conditioned avoidance acquisition (50 massed trials) were examined 68-72 hr after vehicle or pCPA administration in rats which had received control operations or MR lesions two weeks earlier. Only the MR lesion and the MR lesion + pCPA groups evidenced increased open field activity and facilitated two-way avoidance learning. Although the reduction in forebrain 5-HT of the pCPA group (85%) was greater than in the MR lesion group (55%), the pCPA treated animals did not differ from the control group. In the second experiment animals were tested in the open field 24, 48 or 72 hr after pCPA treatment to determine its effects on activity level as a function of the time after injections. No differences between the vehicle and pCPA groups, however, were found. In the third experiment, the effects of pCPA (72 hr postinjection) on the acquisition of an unsignalled one-way avoidance response was examined. MR lesion rats tested in the same apparatus and with the same procedure repeatedly have been shown to be impaired in this task. The pCPA and vehicle animals, however, did not differ. Reduction in 5-HT following electrolytic MR lesions and pCPA administration, thus, produce different behavioral effects. MR lesions, but not pCPA treatment, result in (1) increased activity in a novel environment, (2) facilitated two-way conditioned avoidance learning, and (3) impaired acquisition of an unsignalled one-way avoidance response. These data support earlier studies suggesting that the behavioral effects of electrolytic MR lesions are not due primarily to their disruption of ascending 5-HT pathways. The role of 5-HT in avoidance conditioning and the regulation of activity level, furthermore, remains to be elucidated.

Intra-raphe muscimol induced hyperactivity depends on ascending serotonin projections

Acute microinjections of the GABA agonist, muscimol (100 ng), into either the dorsal (DR) or the median (MR) raphe nucleus of etherized rats induced post-anesthesia hyperactivity as measured in photocell chambers. The increased activity counts seen after MR injections, furthermore, were 4 times greater than those following DR injections. In animals implanted with chronically indwelling cannulae, a muscimol (25-400 ng) dose-response analysis confirmed the greater sensitivity of the MR site. Subsequent experiments thus employed only MR cannulae. The benzodiazepine, chlordiazepoxide, in a subataxic dose (3.8 mg/kg, IP) by itself did not affect activity level, but enhanced the locomotor response to low doses (25-50 ng) of muscimol. Conversely, a sub-convulsant dose of the GABA antagonist, bicuculline (1.1 mg/kg, IP), completely blocked the hyperactivity produced by muscimol (50-100 ng). Bilateral electrolytic destruction of the ventral tegmental nuclei of Gudden produced hyperactivity, but failed to alter the effect of muscimol. Forebrain 5-hydroxytryptamine (5-HT, serotonin) depletion following administration of 5,7-dihydroxytryptamine did not affect baseline activity level, but markedly attenuated the locomotor response produced by intra-MR injections of muscimol. These data suggest that midbrain GABA neurons modulate activity level through a direct action on 5-HT neurons, and indicate that intra-MR muscimol induced hyperactivity depends on intact ascending 5-HT fibers.

Preliminary evidence for methamphetamine-induced behavioral and ocular effects in rat offspring following exposure during early organogenesis

Gravid Sprague-Dawley CD (VAF) rats received 50 mg/kg (d,l)-methamphetamine (MA) HCl (expressed as free base,N=15) or distilled water (N=6) by SC injection × 2/day in a 3 ml/kg volume on embryonic (E) days 7–12. Control rats were pair-fed to MA-exposed dams on days E7–18. No control dams failed to deliver; however, of 15 MA-exposed dams 4 did not deliver (2 died and 2 had completely resorbed litters). One additional MA litter had all the offspring die shortly after birth. There was no difference between groups on offspring postnatal (P) body weight. The offspring exposed prenatally to MA had significantly lower olfactory orientation scores (P9, 11, 13) to their home cage scent. In a test of early activity (P10, 12, 14) the MA-exposed progeny were marginally less active than controls. MA-exposed offspring exhibited hyperreactivity and marginally shortened response latency on a test of acoustic startle (P27). Motor activity showed no differential response in MA treated or control offspring to MA (P63) or fluoxetine challenge (P70). However, the MA offspring were more active than controls with respect to central and side activity during the second week of testing. No group differences were found for performance in a straight swimming channel or on the number of errors committed or latency to escape in a complex (Cincinnati) water maze (P84). Prenatal exposure to MA also induced eye defects (i.e., anophthalmia, microphthalmia and folded retina) in 16.7% of the progeny. However, MA did not effect hippocampal or neostriatal monoamine levels when measured on P28. These data support the hypothesis that MA is behaviorally and ocularly teratogenic to the developing rat and results in functional deficits when compared to offspring of pair-fed controls.

Intra-raphe benzodiazepines enhance rat locomotor activity: Interactions with GABA

Intracranial dose response relationships for the water-soluble benzodiazepines, chlordiazepoxide, flurazepam and midazolam, were performed by injecting the drugs through cannulae chronically indwelling in the median raphe nucleus of male albino rats. Drugs were administered in doses of 0.0, 0.22, 0.44, 0.88 and 1.75 nmole in 0.5 microliters saline. Both midazolam and flurazepam produced hyperactivity which was most prominent within the first 30 minutes post-injection. Flurazepam, furthermore, proved twice as potent as midazolam. Chlordiazepoxide, in contrast, was without effect at any of the doses tested. This observation supports the view that chlordiazepoxide is a pro-drug which must be metabolized to form an active metabolite. In another experiment, animals received either saline or a sub-effective dose (0.22 nmole) of flurazepam or midazolam into the median raphe nucleus 5 minutes prior to either a subeffective dose of muscimol (0.22 nmole) or saline. Only the combinations of a benzodiazepine plus muscimol produced hyperactivity. These combinations, moreover, produced effects as robust as those of a 4-fold higher dose of muscimol alone (0.88 nmole). Other animals received either saline or bicuculline methiodide (0.88 nmole). Bicuculline did not affect activity level, but completely blocked the hyperkinetic effects of muscimol. These data suggest that the hyperactivity effect of intra-raphe muscimol is due to activation of GABA receptors within the midbrain raphe, rather than at distant sites. In addition, the data suggest that the intra-raphe administration of certain benzodiazepines produces hyperactivity by facilitating GABA transmission.

Influence of stimulation of the septal area on visual evoked potentials

Abstract The effects of conditioning stimulation of the septal area or medial thalamus on the amplitude of evoked potentials recorded from the lateral gyrus (VI) of cats were determined. Septal stimulation attenuated whereas stimulation of the central medial thalamic nucleus augmented photically evoked potentials. The cortical response to a single shock to the lateral geniculate body, on the other hand, was enhanced following septal stimulation. As septal stimulation was effective in attenuating photically evoked potentials only when the interval between conditioning and test stimuli was quite large (at least 75 msec) and because of the absence of known direct connections between the septal area and VI, it is probable that the influence of septal stimulation on cortical evoked potentials is multisynaptically mediated. The data were interpreted as suggesting that the septal area is involved in processing visual information. The results were discussed with regard to the origin of the stimulus eliciting the evoked potential and the similar effects of septal and reticular stimulation.

Amyloid-β Injection in Rat Amygdala Alters Tau Protein but Not mRNA Expression

Abstract Previously we demonstrated local and distant changes in tau protein immunoreactivity reminiscent of those seen in Alzheimers disease (AD) following a unilateral injection of amyloid-β (Aβ)25–35 into the rat amygdala. To explore the relevance of these findings to AD, we compared the effects of Aβ1–42 to those of Aβ25–35. Injections of both Aβ1–42 and Aβ25–35 into rat amygdala resulted in increased tau-2 immunolabeling in neurons. To determine whether these alterations were due to changes in the expression of tau, we measured tau protein expression by Western blotting and tau mRNA isoform expression by the reverse transcription-polymerase chain reaction in the amygdala, hippocampus, and cerebellum following a unilateral injection of Aβ25–35 or vehicle into the amygdala. The levels of tau proteins were increased bilaterally in the amygdala of Aβ25–35- compared to vehicle-treated animals 8 and 16 days following treatment. The molecular weights of tau proteins were decreased in the Aβ25–35-treated (59–69 kDa) compared to the vehicle-treated (67–72 kDa) animals 8 days following treatment. There were no changes in tau mRNA expression in any brain region examined. In this model, just as in AD, there is an increase in tau protein levels without a change in tau mRNA expression, suggesting that Aβ peptides may influence tau protein stability in both the rat and the human brain.

Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions ☆

Rats were implanted with cannulae in the median raphe nucleus (MR). 5,7-Dihydroxytryptamine (5,7-DHT) or vehicle was infused either directly through the MR cannula, or bilaterally into the medial forebrain bundle (MFB). The MR 5,7-DHT lesions completely blocked the hyperactivity elicited by injections into the MR of the neurokinin (NK) 3 agonists, DiMe-C7 and senktide, and the NK-2 agonist, neurokinin A. In contrast, the MFB 5,7-DHT lesions did not affect the locomotor hyperactivity produced by intra-MR administration of DiMe-C7 and senktide, but appeared to attenuate the effects of NKA. The data indicate that intra-raphe neurokinin-induced hyperactivity is mediated by 5-HT neurons, and that 5-HT projections to the forebrain may be involved in the behavioral activation induced by intra-raphe neurokinin A administration, but not that induced by intra-MR NK-3 agonists.

Laterality in the histological effects of injections of amyloid-β 25-35 into the amygdala of young Fischer rats

We have observed that single amyloid-beta 25-35 (A beta) injections (5.0 nmol) into the right amygdala of rats produce progressive cytoskeletal and astrogliotic reactions not only within the amygdala, but also in distal brain regions that project to the amygdala. To determine if these effects are potentiated by bilateral injections, we injected A beta (5.0 nmol) into the left and right amygdala of young male Fischer rats. Animals were sacrificed 32 days postoperatively. Bilateral infusions of A beta induced significant neuronal shrinkage, tau-2 neuronal staining, and reactive astrocytosis within the right amygdala and/or hippocampus, compared with vehicle-treated rats. Surprisingly, the same brain regions within the left hemisphere were significantly less affected even though no differences were observed between the left and right amygdala in the size of Congored-positive A beta deposits. Unilateral injections of A beta into the left amygdala led to significant histological changes in the right amygdala and hippocampus, but not in the same brain regions within the left hemisphere. These results suggest a laterality in the histopathological effects of A beta in male Fischer rats. Identification of the cause for the lateralized effect of A beta may prove valuable for understanding the etiology of Alzheimer disease and provide possible therapeutic strategies designed to slow the progression of the disease.

Muscimol injections into the median raphe nucleus increase serum ACTH and corticosterone concentrations via a nonserotonergic mechanism

Midbrain raphe serotonin (5-HT) neurons can influence the pituitary-adrenal axis. The midbrain raphe nuclei also contain a number of non-5-HT neurons, including gamma-aminobutyric acid (GABA) interneurons which can modulate 5-HT neuronal activity. We investigated the effects of intraraphe injections of the GABAA agonist, muscimol, on serum adrenocorticotropin hormone (ACTH) and corticosterone concentrations. Rats were infused with muscimol (0, 25, 50, and 100 ng in 0.5 microliters saline) into the median raphe nucleus (MR). The animals were killed 30 min later, and trunk blood was collected for measurement of serum concentrations of ACTH and corticosterone by radioimmunoassay. Muscimol dose dependently increased plasma concentrations of these two pituitary-adrenal hormones. In order to determine the role of MR 5-HT neurons in these effects, separate groups of implanted animals were infused with either the serotonergic neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT) or ascorbic acid vehicle into the MR. Two weeks later, the animals were infused with muscimol (100 ng in 0.5 microliters) and sacrificed as above. Treatment with 5,7-DHT, which markedly reduced hippocampal concentrations of 5-HT (-83%) and 5-HIAA (-73%), did not block intra-MR muscimol-induced elevations in ACTH and corticosterone. Thus, 5-HT neurons within the MR apparently do not mediate the increased activity of the pituitary-adrenal axis produced by stimulation of MR GABAA receptors.

Effects of amphetamine, food deprivation and current intensity on self-stimulation in the rat

Intracranial self-stimulation (SS) was studied in rats with chronically implanted lateral hypothalamic (LH), substantia nigra (SN), or medial frontal cortex (MF) bipolar electrodes. The effects of Dand L-amphetamine HC1 (0.1, 0.5, 1.0 and 2.0 mg/kg, i.p., as the salt) on SS rate at the 3 electrode sites were compared. The amphetamines were injected at 72-h intervals with each rat receiving each dose (in a random order) of each isomer once, and the effects of the drug on SS rate observed 30-45 min after injection. Both Dand L-amphetamine significantly facilitated LH and SN SS rate at the 0.5, 1.0 and 2.0 mg/kg doses. For each rat in the LH group D-amphetamine had a greater peak facilitatory effect on SS rate than the L isomer. At the SN electrode site the two amphetamine isomers had similar peak facilitatory effects. Neither amphetamine isomer significantly affected MF SS rate. D-Amphetamine resulted in a dose related increase in motor activity, while the same doses of the L isomer resulted in decreased motor activity. Only LH SS rates increased significantly in response to 72-h food deprivation. Increase in current intensity above the level used for amphetamine and food deprivation testing facilitated LH and SN SS rates but did not significantly affect MF SS rate. Currently we are comparing an animals preference for LH vs. MF SS as a function of stimulation intensity and drug administered. These as well as other data suggest that SS is not mediated by a unitary anatomical and/or neurochemical system.